Deans' stroke musings

Changing stroke rehab and research worldwide now.Time is Brain!Just think of all the trillions and trillions of neurons that DIE each day because there are NO effective hyperacute therapies besides tPA(only 12% effective). I have 493 posts on hyperacute therapy, enough for researchers to spend decades proving them out. These are my personal ideas and blog on stroke rehabilitation and stroke research. Do not attempt any of these without checking with your medical provider. Unless you join me in agitating, when you need these therapies they won't be there.

What this blog is for:

Shortly after getting out of the hospital and getting NO information on the process or protocols of stroke rehabilitation and recovery I started searching on the internet and found that no other survivor received useful information. This is an attempt to cover all stroke rehabilitation information that should be readily available to survivors so they can talk with informed knowledge to their medical staff. It's quite disgusting that this information is not available from every stroke association and doctors group.
My back ground story is here:

Tuesday, June 28, 2011

Nicotinergic impact on focal and non-focal neuroplasticity induced by non-invasive brain stimulation in non-smoking humans

I think this says nicotine in patch form helps with neuroplasticity. 32 pages in all.
Nicotine improves cognitive performance and modulates neuroplasticity in brain networks. The
neurophysiological mechanisms underlying nicotine-induced behavioral changes have been
sparsely studied, especially in humans. Global cholinergic activation focuses plasticity in
humans. However, the specific contribution of nicotinic receptors to these effects is unclear.
Henceforth, we explored the impact of nicotine on non-focal neuroplasticity induced by
transcranial direct current stimulation (tDCS) and focal, synapse-specific plasticity induced by
paired associative stimulation (PAS) in healthy non-smoking individuals. Forty eight subjects
participated in the study. Each subject received placebo and nicotine patches combined with one
of the stimulation protocols to the primary motor cortex in different sessions. Transcranial
magnetic stimulation (TMS) - elicited motor evoked potential (MEP) amplitudes were recorded
as a measure of corticospinal excitability until the evening of the second day following the
stimulation. Nicotine abolished or reduced both PAS- and tDCS-induced inhibitory
neuroplasticity. Non-focal facilitatory plasticity was also abolished, whereas focal facilitatory
plasticity was slightly prolonged by nicotine. Thus, nicotinergic influence on facilitatory, but not
inhibitory plasticity mimics that of global cholinergic enhancement. Therefore, activating
nicotinic receptors has clearly discernable effects from global cholinergic activation. These
nicotine-generated plasticity alterations might be important for the effects of the drug on
cognitive function.

Monday, June 27, 2011

Role of transcription factors in neurogenesis after cerebral ischemia

The last line with all the factors listed sure sounds like a dedicated resLinkearcher could work for years on this stuff.


Studies have revealed that the adult mammalian brain has the capacity to regenerate some neurons after cerebral ischemia. And this perspective on neurogenesis adds to the conceptual framework for strategies for the repair of ischemia-induced brain injury, that is, if the effect of ischemia-induced neurogenesis is enhanced, then the recovery of brain function after stroke can be promoted. Neurogenesis is a multistep process that requires the proliferation of neural stem/progenitor cells, migration and that new cells differentiate, survive and integrate into existing neural networks. For that to occur, the same concerted action of various factors is needed, especially transcription factors which regulate the expression of many moleculars and interact with them to promote neurogenesis. This review article gives a brief overview of some transcription factors (NF-κB, Hes, STAT3, AP-1, CREB, HIF1, Pax6, Tcf/Lef, Gli, Sox2, Olig2, Dlx2, TLX, Bmi-1) in ischemia-induced neurogenesis.

The Pessimist's and Optimist's Views of Adult Neurogenesis

I'm not willing to spend $31.50 to find out what these scientists have to say., I wonder if one of my German readers could get the complete article if their government sponsored the research.

The reports by Bonaguidi et al. (in this issue of Cell) and Encinas et al. (in Cell Stem Cell) come to differing conclusions about whether and how the proliferation of radial glia-like stem cells of the adult hippocampus impacts their long-term potential for neurogenesis

Prism Adaptation Therapy Enhances Rehabilitation of Stroke Patients With Unilateral Spatial Neglect:

Its looks like a therapy possibility for spatial neglect. So wLinkho will follow up with trials and get this into vision protocols?


Background and objective. Unilateral spatial neglect (USN) can interfere with rehabilitation processes and lead to poor functional outcome. The purpose of this study was to determine whether prism adaptation (PA) therapy improves USN and functional outcomes in stroke patients in the subacute stage. Methods. A multicenter, double-masked, randomized, controlled trial was conducted to evaluate the effects of a 2-week PA therapy on USN assessed with the Behavioral Inattention Test (BIT), the Catherine Bergego Scale (CBS), and activities of daily living (ADL) as evaluated with the Functional Independence Measure (FIM). A total of 38 USN patients with right-brain damage were divided into prism (n = 20) and control (n = 18) groups. Patients were divided into mild and severe USN groups according to BIT behavioral test (mild ≥ 55 and severe<55). The prism group performed repetitive pointing with prism glasses that induce rightward optical shift twice daily, 5 days per week, for 2 weeks, whereas the control group performed similar pointing training with neutral glasses. Results. The FIM improved significantly more in the prism group. In mild USN patients, there was significantly greater improvement of BIT and FIM in the prism group. Conclusions. PA therapy can significantly improve ADL in patients with subacute stroke.

Rehabilitation, Disability, and Participation Research: Are Occupational Therapy Researchers Addressing Cognitive Rehabilitation After Stroke?

The answer is no. I bolded the pertinent sections.


I reviewed articles published in the American Journal of Occupational Therapy (AJOT) in 2009 and 2010 to assess (1) whether research was published in the practice area of rehabilitation, disability, and participation and (2) the evidence being produced in an underdeveloped subcategory of this practice area: cognitive rehabilitation after stroke. The review revealed one intervention effectiveness study that addressed cognitive rehabilitation poststroke published in the 2-year period. Further analysis of outside repositories of evidence in this area revealed that although some evidence supports rehabilitation approaches for people with cognitive dysfunction after a stroke, little research has been devoted to this practice area. The poststroke cognitive intervention approaches in use have been shown to have little or no effect on improving everyday life activity. Occupational therapy has a key research and practice role with the poststroke population, and occupational therapists should be at the forefront in developing the science to support the effectiveness of their services.

A Real-Time Interactive MIDI Glove for Domicile Stroke Rehabilitation

I wish they at least would show a picture of it and compare it to the saebo-flex and other gloves out there.


Stroke is the leading cause of chronic adult disability in Western countries. After several weeks of inpatient physiotherapy, stroke patients are forced to continue unguided and monotonous therapy at home. Consequently, these patients often lose motivation to continue domicile stroke therapy and therefore do not recover to their potential. An interactive real-time MIDI-Glove was developed with the goal of engaging patients in meaningful, entertaining, and motivating domicile therapy. The MIDI-Glove can also provide a quantitative assessment of progress which provides feedback to both patient and therapist. This paper focuses on two developed MIDI-Glove applications. The first is Musiquant, a computer game which allows an individual to play a sample of a song using the glove and to receive a score based on the performance. The second application allows an individual to play along with a song using a variety of different instruments.

Saturday, June 25, 2011

Transplantation of Flk-1+ human bone marrow-derived mesenchymal stem cells promotes angiogenesis and neurogenesis after cerebral ischemia in rats

You get both new blood vessels and neurons with this one. Now if someone would test it in humans, I'll volunteer.


Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. Although BMSCs-induced angiogenesis is considered important for neurological functional recovery, the neurorestorative mechanisms are not fully understood. We examined whether BMSCs-induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. Adult female rats subjected to 2 h middle cerebral artery occlusion (MCAO) were transplanted with a subpopulation of human BMSCs from male donors (Flk-1+ hBMSCs) or saline into the ipsilateral brain parenchymal at 3 days after MCAO. Flk-1+ hBMSCs-treated rats exhibited significant behavioral recovery, beginning at 2 weeks after cerebral ischemia compared with controls. Moreover, rats treated with Flk-1+ hBMSCs showed increased glucose metabolic activity and reduced infarct volume. Flk-1+ hBMSCs treatment significantly increased the expression of vascular endothelial growth factor and brain-derived neurotrophic factor, promoted angiogenesis, and facilitated cerebral blood flow in the ischemic boundary zone. Further, Flk-1+ hBMSCs treatment enhanced proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone and subgranular zone of the hippocampus. Finally, more NSPCs migrated toward the ischemic lesion and differentiated to mature neurons or glial cells with less apoptosis in Flk-1+ hBMSCs-treated rats. These data indicate that angiogenesis induced by Flk-1+ hBMSCs promotes endogenous neurogenesis, which may cause functional recovery after cerebral ischemia.

Oxytocin stimulates adult neurogenesis even under conditions of stress and elevated glucocorticoids

Someday all these neurogenesis possibilities will be put into a research study and come up decent protocols.


Oxytocin has been linked to social behavior, including social recognition, pair bonding and parenting, but its potential role in promoting neuronal growth has not been investigated. We show here that oxytocin, but not vasopressin, stimulates both cell proliferation and adult neurogenesis in the hippocampus of rats. Oxytocin is also capable of stimulating adult neurogenesis in rats subjected to glucocorticoid administration or cold water swim stress. These findings suggest that oxytocin stimulates neuronal growth and may protect against the suppressive effects of stress hormones on hippocampal plasticity. © 2011 Wiley-Liss, Inc.

Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus

I think I need a scientist to translate this one.


The sequential production of neurons and astrocytes from neuroepithelial precursors is a fundamental feature of central nervous system development. We report that LIM-homeodomain (LIM-HD) transcription factor Lhx2 regulates this transition in the developing hippocampus. Disrupting Lhx2 function in the embryonic hippocampus by in utero electroporation and in organotypic slice culture caused the premature production of astrocytes at stages when neurons are normally generated. Lhx2 function is therefore necessary to suppress astrogliogenesis during the neurogenic period. Furthermore, Lhx2 overexpression was sufficient to suppress astrogliogenesis and prolong the neurogenic period. We provide evidence that Lhx2 overexpression can counteract the instructive astrogliogenic effect of Notch activation. Lhx2 overexpression was also able to override and suppress the activation of the GFAP promoter by Nfia, a Notch-regulated transcription factor that is required for gliogenesis. Thus, Lhx2 appears to act as a “brake” on Notch/Nfia-mediated astrogliogenesis. This critical role for Lhx2 is spatially restricted to the hippocampus, because loss of Lhx2 function in the neocortex did not result in premature astrogliogenesis at the expense of neurogenesis. Our results therefore place Lhx2 as a central regulator of the neuron-glia cell fate decision in the hippocampus and reveal a striking regional specificity of this fundamental function within the dorsal telencephalon.

Friday, June 24, 2011

Evidence from cognitive neuroscience supports action observation as part of an integrated approach to stroke rehabilitation

40 pages trying to explain how observing an action helps stroke rehabilitation. I wonder whether this is still applicable since mental imagery was not found to be helpful;
And where do mirror neurons fit in this possibility?
the 40 page article here:
the first two paragraphs with interesting parts highlighted;
This review of cognitive neuroscience research aims to show
that observing meaningful actions, for example, using a rowing
machine (Fig. 1), can contribute positively to the stroke rehabilitation
Following a cerebrovascular accident, individuals may be left
with chronic motor impairment (e.g. hemiplegia), and cognitive
and psychological disability. Fortunately, recent advances in brain
imaging have led to a greater understanding of the mechanisms of
post-stroke recovery. This knowledge has been essential for optimizing
the efficacy of interventions aimed at promoting motor
recovery. All forms of neural reorganisation are possible following
stroke and include: synaptogenesis; diaschisis, sprouting from
surviving neurons; and recruitment of functionally homologous
(Rossini, Calautti et al., 2003) Therefore, any functional
post-stroke intervention should attempt to enrich and optimise
neural stimulation in order to promote this brain plasticity.
Unfortunately, many traditional therapies have tended to focus on
the recovery of prescriptive physical functions and on the
resumption of generic activities of daily living and, in so doing, have
neglected previously valued activities which have personal
importance to individuals (Cott, Wiles et al., 2007)In my case canoeing. Research that
has attempted to address some of these concerns, by considering
the effectiveness of motor imagery after stroke, has suggested some
promising results (see Page, 2010 for a review). However, a number
of practical and procedural concerns still remain (see Holmes
2007). Until recently, video presented practical problems for the
amateur user; cost, size of equipment, technological understanding
of hardware and editing software were all barriers to its use. The
developments in ubiquitous digital media now allow for the quick
and easy production of high definition images that can address
imagery generation and ability problems, and provide relevant
contextual information. As well as the methodological benefits of
this approach, action observation has also received support from
the neuroscience literature.

Thursday, June 23, 2011

The National Alzheimer’s Project Act (NAPA)

So I wonder what type of stroke association would work on getting an act like this for survivors? And stroke is the 3rd leading cause of death.
What is the National Alzheimer’s Project Act?

The National Alzheimer’s Project Act (Public Law 111-375) requires creation of a national strategic plan to address the rapidly escalating Alzheimer’s disease crisis and will coordinate Alzheimer’s disease efforts across the federal government.

This national strategic framework will include outcome-driven objectives, recommendations, implementation steps and accountability in the fight to overcome Alzheimer’s.

What does the law require?

An annually updated national plan submitted to Congress on how to overcome Alzheimer’s.
Annual recommendations for priority actions to both improve health outcomes for individuals with Alzheimer’s and lower costs to families and government programs.
The annual evaluation of all federally funded efforts in Alzheimer’s research, care and services – along with their outcomes.
The creation of an Advisory Council on Alzheimer’s Research, Care, and Services.

What will the Advisory Council on Research, Care, and Services do?

The Advisory Council will coordinate federal agencies conducting Alzheimer’s-related care, services and research. It will also allow participation in the evaluation and strategic planning process by patient advocates, health care providers, state health departments, Alzheimer’s researchers and health associations.

Participation in the Advisory Council includes the following:

Federal Representation:

Administration on Aging
Agency of Healthcare Research and Quality
Centers for Disease Control and Prevention
Centers for Medicare and Medicaid Services
Department of Veterans Affairs
Food and Drug Administration
Indian Health Service
National Institutes of Health
National Science Foundation
The Surgeon General

Non-Federal Representation (2 each)

Alzheimer’s Caregivers
Alzheimer’s Patient Advocates
Health Care Providers
Researchers with Alzheimer’s Experience
State Health Departments
Voluntary Health Associations

Why is NAPA important?

For too many individuals with Alzheimer’s and their families, the system has failed them, and today we are unnecessarily losing the battle against this devastating disease. The government must make a meaningful commitment to overcome Alzheimer’s.
Alzheimer’s is the 6th leading cause of death in the United States and is the only cause of death among the top 10 in America without a way to prevent, cure or even slow its progression.
By making Alzheimer’s a national priority, we have the potential to create the same success that has been demonstrated in the fights against other diseases. Leadership from the federal government has helped lower the number of deaths from other major diseases such as HIV/AIDS, influenza and pneumonia, and stroke.
NAPA will allow Congress to assess whether the nation is meeting the challenges of this disease for families, communities and the economy. Through its annual review process, NAPA will, for the first time, enable Congress and the American people to answer this simple question: Did we make satisfactory progress this past year in the fight against Alzheimer’s?

Wednesday, June 22, 2011

ForceShoe measures your every move, but won't win fashion awards

Maybe if this is put together with this sock we could actually measure and determine how to correct stroke gaits.
ForceShoe measures your every move, but won't win fashion awards

Developed to help doctors analyze how a stroke patient is using their feet, these shoes have a slew of sensors that measure where the patient is putting pressure as they walk. This data then helps them to develop therapies to assist the patient as they relearn how to walk.

Created by researchers at The University of Twente in the Netherlands, the ForceShoe sends the collected information wirelessly to a computer for analysis, meaning the patient no longer needs to go to a specialized movement laboratory.

Professor Peter Veltink sees plenty of uses beyond rehabilitating stroke patients, and thinks there is great potential for the shoe in sports medicine helping athletes to improve their running and jumping techniques.

I just think they're going to have to come up with a less dorky looking version if they expect LeBron to start wearing them.

Neuroprotective and Ameliorative Actions of Polyunsaturated Fatty Acids Against Neuronal Diseases:

So does this mean I can eat fatty foods?

Neuroprotective and Ameliorative Actions of Polyunsaturated Fatty Acids Against Neuronal Diseases:
Implication of Fatty Acid–Binding Proteins (FABP) and G Protein–Coupled Receptor 40 (GPR40) in Adult Neurogenesis
ABSTRACT: Adult neurogenesis in the mammalian brain is well-known to occur in the subgranular zone of the hippocampus. As the hippocampus is related to learning, memory, and emotions, adult hippocampal neurogenesis possibly contributes to these functions. Adult neurogenesis is modulated by polyunsaturated fatty acids (PUFA) such as docosahexaenoic and arachidonic acids that are essential for normal brain development, maintenance, and function. They are reported to improve spatial learning and memory in rodents and cognitive functions in humans. However, detailed mechanisms of PUFA effects still remain obscure. PUFA are functionally linked with chaperons called fatty acid–binding proteins (FABP). FABP uptake and transport PUFA to different intracellular organelles. Intriguingly, PUFA were determined as ligands for G protein–coupled receptor 40 (GPR40), a cell membrane receptor abundantly expressed in the brain and the pancreas of primates. While the role of GPR40 in pancreatic β-cells is associated with insulin secretion, its role in the brain is not yet clarified presumably because of its absence in the rodent brain. The purpose of this review is to discuss the role of PUFA in adult neurogenesis, considering the role of GPR40 and FABP in the hippocampal neurogenic niche. Here, the authors would like to introduce a PUFA–GPR40 signaling pathway that is specific for the primate brain.

Adult brain neurogenesis and psychiatry: a novel theory of depression

From May 2000, once again proving how poorly stroke research is followed up and transformed into therapeutic practice. If survivors were in charge we would not settle for this pathetic timeline.

Neurogenesis (the birth of new neurons) continues postnatally and into adulthood in the brains of many animal species, including humans. This is particularly prominent in the dentate gyrus of the hippocampal formation. One of the factors that potently suppresses adult neurogenesis is stress, probably due to increased glucocorticoid release. Complementing this, we have recently found that increasing brain levels of serotonin enhance the basal rate of dentate gyrus neurogenesis. These and other data have led us to propose the following theory regarding clinical depression. Stress-induced decreases in dentate gyrus neurogenesis are an important causal factor in precipitating episodes of depression. Reciprocally, therapeutic interventions for depression that increase serotonergic neurotransmission act at least in part by augmenting dentate gyrus neurogenesis and thereby promoting recovery from depression. Thus, we hypothesize that the waning and waxing of neurogenesis in the hippocampal formation are important causal factors, respectively, in the precipitation of, and recovery from, episodes of clinical depression. Molecular Psychiatry(2000) 5, 262-269.

Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects

So you get a twofer, neurogenesis and anti-depressant. Just think you could have your 90 year old parents asking congress to legalize marijuana.
Chronic administration of the major drugs of abuse including opiates, alcohol, nicotine, and cocaine has been reported to suppress hippocampal neurogenesis in adult rats (20–23), suggesting a potential role of hippocampal neurogenesis in the initiation, maintenance, and treatment of drug addiction (13). The recent finding of prominently decreased hippocampal neurogenesis in CB1-knockout mice (24) suggests that CB1 receptor activation by endogenous, plant-derived, or synthetic cannabinoids may promote hippocampal neurogenesis. However, endogenous cannabinoids have been reported to inhibit adult hippocampal neurogenesis (25). Nevertheless, it is possible that exo- and endocannabinoids could differentially regulate hippocampal neurogenesis, as exo- and endocannabinoids act as full or partial agonists on CB1 receptors, respectively (11).
The goal of the present study was to test the hypothesis that the potent synthetic cannabinoid HU210 is able to promote hippocampal neurogenesis, leading to the anxiolytic and antidepressant effects of cannabinoids. We demonstrate here that both HU210 and the endocannabinoid AEA promote proliferation of embryonic hippocampal NS/PCs without significant effects on their differentiation, resulting in more newborn neurons. The effects of HU210 on adult hippocampal neurogenesis were quantified in freely moving rats and were correlated with behavioral testing. We show that 1 month after chronic HU210 treatment, rats display increased newborn neurons in the hippocampal dentate gyrus and significantly reduced measures of anxiety- and depression-like behavior. Thus, cannabinoids appear to be the only illicit drug whose capacity to produce increased hippocampal newborn neurons is positively correlated with its anxiolytic- and antidepressant-like effects.

Other Sections▼

Expression of CB1 receptors in embryonic and adult hippocampal NS/PCs.
In the mammalian brain, the CB1 receptor is one of the most abundant G protein–coupled receptors, accounting for most, if not all, of the centrally mediated effects of cannabinoids (5). We reasoned that if cannabinoids were able to regulate neurogenesis, the NS/PCs capable of producing new neural cells would contain CB1 receptors. We therefore employed CB1 antibody immunocytochemistry, Western blotting, and PCR to examine CB1 protein and gene expression in cultured NS/PCs isolated from the hippocampus of E17 rat embryos. About 95% of the total neurosphere cells labeled with Hoeschst stain were also labeled with both CB1 and nestin (a marker for NS/PCs) antibodies (Figure ​(Figure1A).1A). Some Hoechst-labeled cells in the neurospheres exhibited the shape of glial cells, with small round nuclei, and were CB1 immunoreactive but without nestin staining (Figure ​(Figure1A).1A). The staining of CB1 antibody appears specific for 2 reasons. First, Western blots with the same antibody and cultured NS/PC revealed a strong protein band with the molecular weight of 60 kDa (Figure ​(Figure1B),1B), which corresponds to the CB1 receptor (26). Second, we could not detect the positive immunostaining or 60-kDa protein band using the CB1 antibody preabsorbed with the antigen. Using PCR, we further identified a band of the predicted size (1,440 bp) corresponding to the full encoding region of CB1 (Figure ​(Figure1C),1C), suggesting the presence of CB1 transcripts in NS/PCs. Similar results, i.e., CB1 protein and gene expression, were seen in both second and sixth passages of NS/PCs. We then examined adult naive rats sacrificed 2 hours after receiving a single dose of BrdU to label dividing cells. We found that about 90% of BrdU-stained cells in the SGZ were also doubly labeled with CB1 (Figure ​(Figure1D;1D; n = 3). These results suggest that both embryonic and adult hippocampal NS/PCs express CB1 receptors.
Figure 1

Figure 1
Expression of CB1 receptors in NS/PCs. (A) Coimmunofluorescent staining of CB1 and nestin in cultured hippocampal NS/PCs derived from E17 embryos. Hoechst staining was conducted to reveal the total cultured cells. The arrow indicates the glial-like cells, (more ...)
Increased proliferation of embryonic NS/PCs by HU210 and AEA.
To examine the effects of HU210 on NS/PC proliferation, cultured embryonic NS/PCs were incubated with different concentrations of HU210. With the WST-8 assay, changes in NS/PC proliferation between HU210- and vehicle-treated culture were significant at some concentrations of HU210, as evidenced by significant group effects with 1-way ANOVA (F5,18 = 513.129, P < 0.01). Specifically, when 10 nM to 1 μM of HU210 were added to the culture medium containing the mitogenic growth factors bFGF and EGF, the WST-8 assay showed a significant increase in NS/PC proliferation (Tukey post-hoc tests, P < 0.05); 1 nM of HU210 exerted no significant effects (P = 0.072); 10 μM produced profound toxic effects on cultured NS/PCs (Figure ​(Figure2A).2A). Because HU210 can activate both CB1 and CB2 receptors, we next used the selective CB1 receptor antagonist AM281 to identify the possible involvement of CB1 in the action of HU210 on NS/PC proliferation. Although 1 nM to 1 μM of AM281 alone produced no significant effects on NS/PC proliferation, 10 nM to 1 μM of AM281 blocked the promoting effects of 10 nM to 1 μM of HU210 on NS/PC proliferation (1-way ANOVA for repeated measures, F2,25.713 = 16.792, P < 0.01; pairwise comparisons, HU210-treated cells with or without AM281: P < 0.01) (Figure ​(Figure2A),2A), suggesting that HU210 specifically acts on CB1 receptors to promote NS/PC proliferation. While 10 μM of AM281 alone significantly inhibited NS/PC proliferation (P < 0.01), this concentration of AM281 did not exert significant effects in preventing the lethal effects of 10 μM of HU210 on NS/PCs (Figure ​(Figure2A),2A), indicating that the lethal effects of 10 μM of HU210 on NS/PC cells were caused nonspecifically or by another receptor.
Figure 2

Figure 2
Effects of the cannabinoid HU210 on proliferation of cultured hippocampal NS/PCs. (A) In the WST-8 assay, incubation of NS/PCs with 10 nM to 1 μM of HU210 for 48 hours significantly promoted NS/PC proliferation, which was blocked by the CB1 receptor (more ...)
To confirm the effects of 10 nM to 1 μM of HU210 on promoting NS/PC proliferation as previously assessed by the WST-8 assay, the BrdU incorporation assay was used. It measures cell proliferation by detecting dividing cells. Similar to the results of the WST-8 assay, 1-way ANOVA showed significant group effects (F5,18 = 176.004; P < 0.01); Tukey post-hoc tests revealed that 10 nM to 1 μM of HU210 significantly increased NS/PC proliferation (P < 0.05), which was blocked by 10 nM to 1 μM of the selective CB1 receptor antagonist AM281 (1-way ANOVA for repeated measures, F2,36 = 19.081, P < 0.01; pairwise comparisons, HU210-treated cells with or without AM281: P < 0.01) (Figure ​(Figure22B).
To determine the effects of the endogenous cannabinoid AEA on NS/PC proliferation, cultured NS/PCs were incubated with different concentrations of AEA. The WST-8 assay showed significant group effects with 1-way ANOVA (F5,18 = 61.585, P < 0.01). Tukey post-hoc tests further showed that 1 μM to 10 μM of AEA significantly increased NS/PC proliferation (P < 0.05) in the presence of bFGF and EGF; 100 μM produced toxic effects (Figure ​(Figure22C).
To explore the possibility of whether HU210 itself is able to produce mitogenic effects, we further examined NS/PC proliferation by adding different concentrations of HU210 to the culture medium with or without the mitogenic growth factors bFGF and EGF. When bFGF and EGF were absent from the culture medium, a significant overall change in NS/PC proliferation was observed following HU210 application (F5,30 = 219.076, P < 0.01) (Figure ​(Figure2D).2D). Specifically, 10 nM to 1 μM of HU210 without growth factors produced significant mitogenic effects on NS/PCs (Tukey post-hoc tests, P < 0.05), whereas 10 μM of HU210 killed the cells. Similar results were observed in the control culture when different concentrations of HU210 were added to the culture medium containing the mitogenic growth factors (F5,30 = 194.429, P < 0.01; Tukey post-hoc tests, P < 0.05) (Figure ​(Figure2D).2D). Nevertheless, the basal proliferation levels with bFGF and EGF were significantly higher than those without bFGF and EGF (1-way ANOVA for repeated measures, F1,30 = 214.703, P < 0.01; pairwise comparisons: P < 0.01) (Figure ​(Figure22D).
Intracellular signaling involved in HU210-induced NS/PC proliferation.
To investigate the mechanisms underlying the action of HU210 on NS/PC proliferation, we examined the intracellular signaling pathways. CB1 receptor stimulation activates Gi/o or Gs proteins (27, 28). To examine whether Gi/o protein mediates the effects of HU210, we added pertussis toxin, a selective blocker for Gi/o protein activation, to the culture medium 4 hours prior to HU210 treatment. Again, 10 nM to 1 μM of HU210 significantly increased NS/PC proliferation (1-way ANOVA, F5,18 = 880.629, P < 0.01; post-hoc tests, P < 0.01 between control and each of the 3 concentrations of HU210), which was completely blocked by 100 ng/ml of pertussis (1-way ANOVA for repeated measures, F1,18 = 41.64, P < 0.01; pairwise comparisons, HU210-treated cells with or without pertussis: P < 0.01) (Figure ​(Figure2E).2E). It has been shown that HU210 activates Gs proteins when Gi/o proteins are inhibited by pertussis toxin (27). Therefore, to determine whether the blockade effects of HU210-induced NS/PC proliferation following pertussis treatment is achieved by activation of Gs proteins, we examined the effects of cholera toxin, a Gs protein activator, on NS/PC proliferation. Incubation of NS/PCs with 1 mg/ml of cholera toxin stimulated about 14-, 80-, 90-, and 13-fold increase in cAMP accumulation in NS/PCs 0.5, 1, 2, and 24 hours after the addition of cholera toxin; cAMP production returned to the basal levels 48 hours after cholera toxin (1-way ANOVA, F5,18 = 93.341, P < 0.01) (Figure ​(Figure2F).2F). These results indicate the effective activation of Gs proteins in NS/PCs by cholera toxin. However, there was no significant change in NS/PC proliferation 0.5, 1, 2, 24, and 48 hours after the addition of cholera toxin (1-way ANOVA, F5,18 = 76.562, P = 0.86) (Figure ​(Figure2G).2G). These results together suggest the involvement of Gi/o proteins, but not Gs proteins, in HU210-induced NS/PC proliferation.
Since Gi/o protein activates PI3K/Akt and ERK signaling (29), which are well known to play an important role in cell growth and cell death, we studied whether HU210 could activate Akt and ERK1/2. There was no significant change in phosphorylation of phospho-Akt during the first 1 hour after HU210 application (F4,10 = 1.693, P = 0.228) (Figure ​(Figure3A),3A), indicating that the PI3K/Akt signaling pathway is not involved in the action of HU210 on NS/PC proliferation. In contrast, changes in phosphorylation of phospho-ERK1/2 (pERK1/2) during the first 1 hour after HU210 application were dramatic at specific time points, as shown by 1-way ANOVA (with growth factors, F4,15 = 33.698, P < 0.01; without growth factors, F4,15 = 23.513, P < 0.01). As early as 5 minutes after addition of HU210 to culture medium with (Figure ​(Figure3B)3B) or without bFGF and EGF (Figure ​(Figure3C),3C), a 2.5-fold increase in phosphorylation of pERK1/2 was observed (P < 0.05). At 15 minutes after HU210 application, phosphorylation of pERK1/2 reached the peak level, which was about a 4-fold (with growth factors) or 7-fold increase (without growth factors) relative to control (P < 0.01). By 60 minutes after addition of HU210, phosphorylation of pERK1/2 either significantly decreased (P < 0.05) (Figure ​(Figure3B)3B) or returned to the pretreatment level (Figure ​(Figure3C).3C). We did not observe any significant changes in the total ERK1/2 during the first 1 hour after HU210 application. Thus, the significant increase in pERK1/2 in this period suggests an important involvement of ERK signaling pathway in the action of HU210 in promoting NS/PC proliferation. This hypothesis was supported by further experiments in which U0126, a specific inhibitor of the ERK pathway, was employed. Figure ​Figure3D3D shows an overall significant difference in pERK1/2 phosphorylation after application of vehicle or 100 nM of HU210 with or without 10 μM of U0126 (F3,8 = 60.769, P < 0.01). Specifically, HU210 profoundly increased phosphorylation of pERK1/2 (P < 0.01), which was almost completely blocked by U0126 (P < 0.01). A parallel experiment demonstrated that U0126 blocked the promoting effects of 100 nM of HU210 on NS/PC proliferation (1-way ANOVA for repeated measures, F1,17 = 6.356, P < 0.05; pairwise comparisons, HU210-treated cells with or without U0126: P < 0.05) (Figure ​(Figure33E).
Figure 3

Figure 3
Effects of the cannabinoid HU210 on PI3K/Akt and ERK signaling in cultured hippocampal NS/PCs. (A) There was no significant change in pAkt or actin in NS/PCs within the first hour after addition of 100 nM of HU210 to culture medium. (B) Application of (more ...)
HU210 and AEA do not affect neuronal differentiation of cultured NS/PCs.
To examine the effects of HU210 on neuronal differentiation of cultured NS/PCs, neurospheres were dissociated, plated, and cultured in the medium containing bFGF and EGF for 1 day and then in another medium containing different concentrations of HU210 without bFGF or EGF for 8 days. After fixation, immunofluorescence staining was performed using antibodies against the neuronal marker β-tubulin III (TuJ1), followed by Hoechst staining that detects all the cultured cells. Cell counting revealed no significant difference among the ratios of TuJ1-labeled neurons and Hoechst-labeled total cells following treatment with vehicle or 10 nM, 100 nM, or 1 μM of HU210 (1-way ANOVA, F4,20 = 3.307, P = 0.324) (Figure ​(Figure4),4), suggesting that HU210 exerts no significant effects on neuronal differentiation of cultured NS/PCs. Similarly to HU210, AEA (1 and 5 μM) did not produce significant effects on neuronal differentiation of cultured NS/PCs (1-way ANOVA, F2,9 = 0.177, P = 0.840) (Figure ​(Figure44B).
Figure 4

Figure 4
Effects of HU210 and AEA on neuronal differentiation of cultured hippocampal NS/PCs. (A) Incubation of NS/PCs with the culture medium containing either vehicle or 100 nM of HU210 without growth factors for 8 days produced similar density of neurons (pink (more ...)
Increased hippocampal cell proliferation following HU210 treatment in adult rats.
BrdU labeling of dividing cells was used to test the acute effects of HU210 treatment on cell proliferation in adult hippocampus. Adult rats received a single dose of vehicle, AM281 (3 mg/kg, i.p.), or HU210 (25 or 100 μg/kg, i.p.), followed 2 hours later by BrdU administration and then perfusion 1 day later. BrdU-labeled cells showed fusiform or irregular shape and were clustered or aggregated in the SGZ (Figure ​(Figure5A)5A) throughout the whole hippocampus in all rats examined. Cell counting revealed no significant change in the number of BrdU-positive cells in the SGZ among rats treated with vehicle, AM281, or HU210 (1-way ANOVA, F3,16 = 52.784, P = 0.58; n = 5) (Figure ​(Figure5B).5B). We then examined the effects of chronic HU210 injection on cell proliferation in adult hippocampus. Two hours after receiving the last dose of twice-daily injections of vehicle, AM281 (3 mg/kg, i.p.), or HU210 (25 or 100 μg/kg, i.p.) for 10 days, adult Long-Evans rats received BrdU administration and then were perfused 1 day later. Immunohistochemical staining showed an apparent increase in the density of BrdU-labeled cells in the SGZ following chronic administration of 100 μg/kg of HU210 (Figure ​(Figure5C).5C). One-way ANOVA revealed a significant overall difference in the mean ± SEM number of BrdU-positive cells in the SGZ (F3,16 = 11.504, P < 0.001; n = 5) (Figure ​(Figure5D).5D). Tukey post-hoc test showed a significant increase (about 40%) in the number of BrdU-labeled cells following 100 μg/kg of HU210 (P < 0.05) but not 25 μg/kg of HU210 (P = 0.979), relative to vehicle (Figure ​(Figure5D).5D). AM281 injection seemingly decreased the number of BrdU-positive cells in the SGZ, but there was no significant difference relative to control (P = 0.099).
Figure 5

Figure 5
Effects of HU210 treatment on cell proliferation in the dentate gyrus in adult rats (n = 5–7 rats in each group). Cell proliferation was assessed by BrdU labeling of dividing cells. (A) Representative microphotographs of the dentate gyrus (more ...)
Increased newborn hippocampal neurons following chronic HU210 treatment in adult rats.
A recent study has demonstrated that newborn neurons in the dentate granule cell layer that had survived 4 weeks were stably integrated into the granule cell layer (30). To examine the survival, migration, and differentiation of HU210-induced newborn cells in the SGZ, we injected rats twice daily with HU210 (100 μg/kg, i.p.), AM281 (3 mg/kg), or vehicle for 10 days, followed 12 hours later by 4 BrdU injections at 12 hours intervals. One month after the last HU210, AM281, or vehicle injection, the majority of BrdU-labeled cells migrated and dispersed into the granule cell layer and showed size and morphology indistinguishable from both their neighboring granule neurons and from different treatment (Figure ​(Figure6A).6A). The number of BrdU-labeled dentate cells in HU210-treated rats was significantly higher than that in vehicle-treated rats (Student’s t test, P < 0.01; n = 5) (Figure ​(Figure6B),6B), indicating that most of chronic HU210–induced newborn cells survived. Immunofluorescence staining revealed that HU210- and vehicle-treated rats exhibited a similar proportion of BrdU/neuronal nuclear antigen (BrdU/NeuN) double-labeling cells to the total BrdU-labeled cells (Student’s t test, P = 0.977) (Figure ​(Figure6C),6C), suggesting that chronic HU210-induced newborn cells in the SGZ have neuronal differentiation ratio similar to that of vehicle-induced newborn cells in the SGZ. Nevertheless, because chronic HU210 treatment significantly increased the number of BrdU-labeled newborn cells in the dentate gyrus (Figure ​(Figure6B),6B), the total number of newborn neurons doubly labeled with BrdU/NeuN in the dentate gyrus also significantly increased following chronic HU210.
Figure 6

Figure 6
Fate and migration of BrdU-labeled cells in the dentate gyrus following chronic HU210 treatment. After receiving twice-daily injections of vehicle or 100 μg/kg of HU210 for 10 days, rats were given 4 BrdU injections, followed 1 month later by (more ...)
No hippocampal neuronal death following chronic HU210 treatment in adult rats.
Ample evidence has illustrated the increased hippocampal neurogenesis following ischemia, epileptic status, enriched environment, or exercise (15). It is therefore possible that increased hippocampal neurogenesis following chronic HU210 treatment in adult rats may result from the toxic effects of chronic HU210 treatment on hippocampal neurons. To explore this possibility, we examine the total number of the dentate granule and CA3 pyramidal neurons following twice-daily injections of HU210 (100 μg/kg) for 10 days. As depicted in Figure ​Figure7A,7A, HU210-treated rats did not show detectable loss of NeuN-immunopositive neurons in the hippocampus, relative to naive control rats. Stereological cell counting confirmed that no significant difference in the total number of the dentate granule cells (F1,4 = 1.443, P = 0.782) and CA3 pyramidal neurons (F1,4 = 5.099, P = 0.553) between naive and HU210-treated rats (Figure ​(Figure7B).7B). These results, however, do not exclude the possibility that some of NeuN-stain neurons following chronic HU210 treatment shown in Figure ​Figure7A7A are dying. Accordingly, we used TUNEL stain and Fluoro-Jade B stain to examine the degenerating hippocampal neurons (31) in rats receiving chronic HU210 treatment, with the naive rats as negative control and kainic acid–treated rats as positive control (31). We failed to detect any TUNEL- or Fluoro-Jade B–stained degenerating cells throughout the whole hippocampus in both naive rats and HU210-treated rats, whereas kainic acid–injected rats showing epileptic status exhibited numerous dying cells in the CA3 pyramidal cell layer and even dentate granule cell layer (Figure ​(Figure7,7, C and D).
Figure 7

Figure 7
Effects of chronic HU210 on neuronal survival. (A) Both naive control rats and rats receiving twice-daily injections of HU210 (100 μg/kg) for 10 days showed similar density of NeuN-stained neurons in the dentate granule cell layer and CA3 pyramidal (more ...)
Anxiolytic and antidepressant effects of chronic HU210.
Two recent studies employing novelty-suppressed feeding (NSF) tests and forced swimming test (FSTs) as measures of anxiety and depression have shown that chronic treatment with the antidepressant fluoxetine produced anxiolytic and antidepressant effects (18, 19), and the anxiolytic effects are likely achieved by promoting hippocampal neurogenesis (18). Therefore, we employed the same behavioral tests to examine the effects of chronic HU210 treatment on measures of anxiety and depression. Rats received twice-daily injections of vehicle, AM281 (3 mg/kg), or HU210 (100 μg/kg) for 10 days, followed 12 hours later by 4 BrdU injections at 12-hour intervals. Rats were subjected to behavioral testing 1 month later, based on the recent finding that hippocampal newborn neurons need 4 weeks to become functional (32). In the NSF test, 1-way ANOVA showed an overall significant difference in the latency to eat in the novel environment among the 3 groups of rats deprived of food for 48 hours (F2,20 = 8.187, P < 0.01). As shown in Figure ​Figure8A,8A, relative to vehicle treatment, chronic HU210 (but not AM281) treatment significantly reduced the latency to eat food in the novel environment (P < 0.01). However, when returned to their home cages immediately after the test, rats receiving vehicle, chronic AM281, and chronic HU210 showed no significant difference in the latency to eat food (F2,20 = 0.276, P = 0.762) (Figure ​(Figure8A)8A) or the amount of food consumed (F2,20 = 0.839, P = 0.447). In the FST, there was an overall significant difference in the duration of immobility among vehicle-, AM281-, and HU210-treated rats (F2,19 = 4.441, P < 0.05). Post-hoc test revealed that HU210 (but not AM281) significantly decreased immobility (P < 0.05) (Figure ​(Figure8B),8B), whereas neither AM281 nor HU210 produced significant effects on the number of rats climbing in the first 5 minutes in the pretest sessions of the FST (F2,19 = 7.552, P = 0.887) (Figure ​(Figure8C).8C). Rats were killed for immunohistochemical staining after behavioral tests. The majority of BrdU-positive cells in vehicle-, AM281-, or HU210-treated rats were located in the granule cell layer, suggesting that they became granule neurons. Cell counting revealed an overall significant difference in the number of BrdU-stained cells in the dentate gyrus (F2,19 = 3.896, P < 0.05). Post-hoc test showed results similar to those in Figure ​Figure5D:5D: namely, relative to vehicle-treated rats, HU210-treated rats displayed a significant increase (P < 0.05) in the number of BrdU-positive cells in the dentate gyrus, whereas AM281-treated rats exhibited no significant difference (P = 0.165). Thus, these data together suggest that chronic HU210 treatment promoted hippocampal neurogenesis and exerted anxiolytic- and antidepressant-like effects.
Figure 8

Figure 8
Effects of chronic HU210 on the NSF test, the FST, and cell proliferation in the dentate gyrus. After receiving chronic vehicle, AM281, or HU210 injections for 10 days, rats were injected with BrdU to label dividing cells, followed 1 month later by behavioral (more ...)
Association of hippocampal neurogenesis with anxiolytic- and antidepressant-like effects of chronic HU210.
To determine the relationship between hippocampal neurogenesis and anxiolytic- and antidepressant-like effects produced by chronic HU210, we examined the effects of a selective destruction of the hippocampal neural stem cells on the behavioral effects of chronic HU210. During the course of receiving chronic HU210 injections, 1 group of Long-Evans rats received two 5-Gy doses of x-rays confined to a limited brain region including the hippocampus, as previously described (18). Four BrdU injections with 12-hour intervals were given after the last HU210 injection. Hippocampal irradiation produced a prominent decrease in the number of BrdU-positive cells in the SGZ (F2,12 = 6.011, P < 0.01) (Figure ​(Figure8D)8D) and a blockade of chronic HU210–induced anxiolytic-like effects (F2,12 = 4.209, P < 0.05) (Figure ​(Figure8E)8E) and antidepressant-like effects (F2,12 = 9.100, P < 0.05) (Figure ​(Figure8F)8F) without significant effects on amount of food consumed when rats were returned to their home cages (F2,12 = 2.376 P = 0.502) (Figure ​(Figure8E)8E) and climbing times (F2,12 = 9.113, P = 0.624). Because two 5-Gy doses of x-rays were not found to alter the morphology and function of mature neurons in the hippocampus, hypothalamus, and amygdala (18), our results together suggest that chronic HU210 treatment reduced anxiety and depression, likely via promoting hippocampal neurogenesis.

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Natural selection has conserved cannabinoid receptors in various vertebrates and invertebrates that have been evolutionarily separate for 500 million years (33), indicating the importance of cannabinoid receptors to life. A recent study has shown CB1-immunoreactive newborn neurons in the adult rat hippocampus 1 week after BrdU injection (24). Here we have observed that approximately 95% of cultured neurosphere cells were doubly labeled with CB1 and nestin, a marker for NS/PCs. Western blotting and PCR further showed the expression of CB1 protein and gene in NS/PCs. We also detected cells doubly stained with CB1 and BrdU in the SGZ of adult rats that were sacrificed 2 hours after receiving a single dose of BrdU. This time interval allowed us to label mitotically active cells (i.e., NS/PCs) in the hippocampal SGZ, as they have a doubling time of 11–25 hours (14). Therefore, this study provides the first evidence suggesting that both embryonic and adult hippocampal NS/PCs express CB1 receptors.
Accordingly, we hypothesized that cannabinoids could regulate proliferation of hippocampal NS/PCs by acting on CB1 receptors. This hypothesis is supported by our subsequent findings that both the synthetic cannabinoid HU210 and endocannabinoid AEA profoundly promoted embryonic hippocampal NS/PC proliferation, and the effects of HU210 could be blocked by the selective CB1 receptor antagonist AM281. Furthermore, we discovered the mitogenic effects of HU210 on cultured NS/PCs in the absence of FGF-2 and EGF in the culture medium, thus excluding the possibility that HU210 induces NS/PC proliferation via indirect action through FGF-2 and EGF and reiterating the direct action of HU210 on CB1 receptors on the cultured NS/PCs. We next observed that HU210 promoted NS/PC proliferation, likely via Gi/o protein activation and subsequent ERK signaling. Although both HU210 and AEA exerted no significant effects on neuronal differentiation of NS/PCs, they significantly increased NS/PC proliferation, leading to increased total number of newborn neurons. Similar results were also obtained in freely moving adult rats. That is, chronic, but not acute, HU210 significantly increased the number of newborn hippocampal neurons in adult rats by promoting NS/PC proliferation but not differentiation. We also provided evidence indicating that the promoting effects of chronic HU210 treatment on adult hippocampal neurogenesis are not the outcome of hippocampal neuronal death, as we did not detect neuronal loss or dying hippocampal neurons following chronic HU210 injection. Overall, these data support the idea that cannabinoids are able to promote embryonic and adult hippocampal neurogenesis via the CB1 receptors in the NS/PCs.
Our findings of cannabinoid-induced increase in hippocampal neurogenesis are in agreement with the recent observation that CB1 receptor–knockout mice display profound suppression of hippocampal neurogenesis (24). However, our observation that both HU210 and AEA did not affect neuronal differentiation of embryonic hippocampal NS/PCs is different from Rueda et al.’s study showing that endocannabinoids and HU210 inhibited neuronal differentiation of cultured embryonic cortical and human NS/PCs and PC12 cells stably transfected with human CB1, which was blocked by CB1 receptor antagonist (25). These differing results may be due to the differing effects of cannabinoids on embryonic cortical and hippocampal NS/PCs. In in vivo experiments, Rueda et al. demonstrated that chronic administration of endogenous cannabinoid for 2 weeks increased the number of newborn (BrdU-immunopositive), non-neuronal (NeuN-immunonegative) cells in the rat dentate gyrus without affecting the total number of BrdU-labeled cells (25), which was interpreted as evidence for a CB1-mediated impairment in neurogenesis. We observed, however, a significant increase in the hippocampal newborn neurons following twice-daily HU210 injection for 10 days. The differing regulatory effects of endocannabinoid shown in Rueda et al.’s study and exocannabinoid HU210 shown in this study on hippocampal neurogenesis may be produced by the different pharmacology of exo- and endocannabinoids in the brain, i.e., the full and partial agonist actions of exo- and endocannabinoids on CB1 receptors, respectively (11); or the different intracellular signaling pathways induced by exo- and endocannabinoids as speculated by Martin et al. (34); or the opposing effects induced by high and low doses of exocannabinoids (35) and endocannabinoids (36). In fact, some studies have shown that exo- and endocannabinoids have differential or opposing effects in many areas, including nociception (37), the vascular system (38), and epilepsy (39).
Following the observation that chronic HU210 treatment promoted neurogenesis in the dentate gyrus, we wondered whether chronic HU210–induced newborn neurons are of functional significance. Given the recent findings that chronic fluoxetine treatment produced antidepressant and anxiolytic effects (18, 19) and the anxiolytic effects are likely achieved by promoting hippocampal neurogenesis (18), we hypothesized that chronic HU210–induced hippocampal neurogenesis may also correlate with anxiolytic and antidepressant effects. Our subsequent experiments supported this hypothesis. After 1 month of chronic HU210 treatment, rats deprived of food for 48 hours showed significantly reduced latency to eat food in a novel environment, suggesting that chronic HU210 treatment exerted anxiolytic effects. These results are consistent with a recent study showing that once-daily injections of the cannabinoid receptor agonist CP55,940 for 11 days reduced anxiety in the elevated plus-maze test performed 30 days after the last CP55,940 injection (40). Chronic HU210–induced shortened latency to eat food in the novel environment is unlikely due to the well-known effects of HU210 on appetite (1), because chronic HU210 treatment produced no significant effects on the latency to eat food or the amount of food consumed when rats were returned to the familiar environment of their home cages immediately after the test. One week after undergoing NSF testing, the same rats receiving chronic HU210 treatment showed a significantly reduced duration of immobility in the FST, indicating that chronic HU210 also exerts antidepressant effects. Because acute cannabinoid treatment profoundly affects motor function of humans and animals (1, 10), chronic HU210–induced shortened immobility in the FST may be produced by its action in changing the motor activity of rats. This is unlikely, however, as we observed no significant difference in the number of rats climbing (41, 42) among HU210-, AM281-, and vehicle-treated groups in the first 5 minutes of the pretest sessions of the FST. The anxiolytic- and antidepressant-like behavioral changes in rats 1 month after chronic HU210 treatment are unlikely to have been produced by the cannabinoid withdrawal effects, since, as shown in our recent study (8), rodents receiving chronic cannabinoid would display detectable cannabinoid withdrawal syndrome only after administration of CB1 receptor antagonists. Finally, the same rats with reduced measures of anxiety and depression following chronic HU210 treatment showed significantly increased numbers of BrdU-labeled cells within the dentate granule cell layer. These overall results thus confirmed our above-described novel findings that chronic HU210 treatment significantly increased newborn neurons in the hippocampal dentate gyrus (Figure ​(Figure6).6). The lack of statistically significant effects produced by the CB1 antagonist AM281 on both hippocampal neurogenesis and behavioral testing suggests that daily temporary blockade of CB1 receptors is not strong enough to affect hippocampal neurogenesis and the regulation of anxiety or depression.
Multiple classes of antidepressant drugs increase hippocampal neurogenesis in a chronic but not acute time course, which corresponds to the therapeutic time course necessary for effects (12, 43). Conversely, cell proliferation is decreased in animal models of depression or stress and anxiety paradigms (12, 43). Further evidence supporting the association of hippocampal neurogenesis with mood and anxiety disorders comes from a recent study. In this study the disruption of antidepressant-induced hippocampal neurogenesis by x-irradiation of a restricted mouse brain region containing the hippocampus blocked anxiolytic effects of several antidepressants (18). We observed similar results in the present study that x-irradiation of a brain region containing the hippocampus blocked both the adult hippocampal neurogenesis and anxiolytic- and antidepressant-like effects of chronic HU210. Thus, all these lines of evidence support the notion that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects via promoting hippocampal neurogenesis.
It has been shown that acute, high doses of CB1 agonists or cannabinoids produced anxiety-like effects in rats (44–49) or depression-like effects in mice (50, 51). We observed here that chronic administration of high, but not low, doses of HU210 exerts anxiolytic- and antidepressant-like effects. To make things more complicated, acute, low doses of cannabinoids have been found to induce anxiolytic-like effects in rodents (44, 49, 52, 53). These complicated effects of high and low doses of acute and chronic exposure to cannabinoids may explain the seemingly conflicting results observed in clinical studies regarding the effects of cannabinoid on anxiety and depression (3, 4, 10).
In summary, since adult hippocampal neurogenesis is suppressed following chronic administration of opiates (20), alcohol (21), nicotine (22), and cocaine (23), the present study suggests that cannabinoids are the only illicit drug that can promote adult hippocampal neurogenesis following chronic administration. Increased hippocampal neurogenesis appears to underlie the mechanism of anxiolytic- and antidepressant-like effects produced by a high dose of chronic HU210 treatment. The opposing effects of high doses of acute and chronic cannabinoids, together with the anxiolytic-like effects caused by a low dose of cannabinoids, may finally explain discrepancies in the clinical study literature regarding the effects of cannabinoid on anxiety and depression.

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All procedures were in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the University of Saskatchewan Animal Care Committee.
Primary NS/PC culture.
NS/PCs were isolated and propagated using a neurosphere method developed by Reynolds and Weiss (54) and modified by Gritti et al. (55). Hippocampi were dissected under a stereomicroscope from E17 Long-Evans rat embryos into HBSS without calcium or magnesium (Invitrogen Corp.). The tissues were then cut into small pieces, digested with 0.05% trypsin/0.53 mM EDTA (Sigma-Aldrich) for 10 minutes at 37°C and triturated with a fire-polished pipette into individual cells. The cells were collected by centrifugation, resuspended in DMEM/F12 medium (1:1 mixture) (Invitrogen Corp.), and gently forced through a 41-μm nylon net filter (Millipore) to form a suspension of disaggregated cells. The disaggregated cells were seeded into uncoated T25 culture flasks (TPP) at a density of 1 × 105 viable cells/ml in DMEM/F12 medium supplemented with B27 supplement (Invitrogen Corp.), 20 ng/ml FGF-2 (Chemicon International), and 20 ng/ml EGF (Invitrogen Corp.). The cells were maintained in a humidified incubator at 37°C with 95% atmospheric air/5% CO2, grown as free-floating aggregates (neurospheres), and passaged every 5–6 days. All experiments were carried out using the second passage of NS/PCs.
NS/PC proliferation assay.
Single-cell suspensions were prepared from neurospheres by enzymatic dissociation with trypsin-EDTA. The cells were then allowed to pass through a 40-μm nylon net filter and plated in 96-well microplates at a density of 1 × 105 cells/ml, followed 12 hours later by the addition of reagents including 0, 1 nM, 10 nM, 100 nM, 1 μM, or 10 μM of HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] (dissolved in 0.1% [vol/vol] of DMSO; Tocris Bioscience) or AM281 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide] (dissolved in 0.1% [vol/vol] of DMSO; Tocris Bioscience), or 0, 10 nM, 100 nM, 1 μM, 10 μM, or 100 μM of AEA [N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (dissolved in 0.1% [vol/vol] of ethanol; Sigma-Aldrich). The concentration range of HU210, AM281, and AEA was determined according to our pilot experiments. In separate experiments, 100 ng/ml of pertussis (Sigma-Aldrich) or 10 μM of U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene] (Cell Signaling Technology) were added 4 hours and 1 hour, respectively, prior to the addition of different concentrations of HU210. The concentrations of pertussis and U0126 were chosen based on the previous findings that these concentrations effectively inhibited Gi/o protein and ERK signaling in cultured NS/PCs (56, 57). After incubating the microplate at 37°C in 5% CO2/95% air for 48 hours, cell proliferation was measured using the WST-8 assay with Cell Counting Kit-8 (Dojindo Molecular Technologies Inc.) or BrdU incorporation assay with Cell Proliferation ELISA BrdU kit (Roche Diagnostics Corp.). In additional experiments, 1 mg/ml of cholera toxin (Sigma-Aldrich) was added, and 0, 0.5, 1, 2, 24, or 48 hours later, the culture medium containing cholera toxin was replaced with the regular culture medium. Cell proliferation was measured 48 hours after addition of cholera toxin using the WST-8 assay. The concentration of cholera toxin was determined after a pilot experiment.
WST-8 assay.
The number of viable cells was estimated using the WST-8 assay, which provides effective and reproducible determination of the proliferative activity of NS/PCs (58). WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] is reduced by the mitochondrial enzyme NAD-dependent succinate dehydrogenase to form a colored formazan product, which is soluble in the culture medium. The amount of formazan dye generated by the activity of the dehydrogenases in cells is known to be directly proportional to the number of living cells. To measure the proliferative activity of NS/PCs in 96-well microplates, 10 μl of the Cell Counting Kit solution were added into each well, followed by incubation of the microplates at 37°C in 5% CO2/95% air for 5 hours. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices) with a reference wavelength of 650 nm.
BrdU incorporation assay.
The cells were labeled with 10 μM of BrdU and incubated at 37°C in 5% CO2/95% air for 4 hours. After centrifugation at 300 g for 10 minutes, the labeling medium was removed by aspiration and the cells were dried. Then, cellular DNA was denatured by adding 200 μl of FixDenat solution into each well, and the microplates were incubated at room temperature for 30 minutes. After removal of the FixDenat solution, 100 μl of anti-BrdU antibody conjugated with peroxidase solution was added to each well, followed by incubation of microplates at room temperature for 90 minutes. After 3 washes with washing solution, 100 μl of substrate solution was added to each well, and the microplates were incubated at room temperature for 40 minutes. The reaction was then stopped by adding 25 μl of 1 M H2SO4 solution into each well. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices) with a reference wavelength of 650 nm.
Measurement of cAMP levels in cultured NS/PCs.
NS/PCs were incubated with 1 mg/ml of cholera toxin for 0, 0.5, 1, 2, 24, or 48 hours, followed by preparation of samples for the measurement of cAMP production using the Direct cAMP Enzyme Immunoassay Kit (Sigma-Aldrich) according to manufacturer’s manual.
Immunocytochemistry for cultured NS/PCs.
To detect CB1 receptor in cultured hippocampal NS/PCs, neurospheres were plated on 12-mm glass coverslips coated with 15 μg/ml of poly-L-ornithine and were cultured in DMEM/F12 medium supplemented with B27 supplement, 20 ng/ml FGF-2, and 20 ng/ml EGF for 24 hours. Cells on coverslips were fixed with 4% paraformaldehyde, rinsed in PBS, and blocked with 5% normal goat serum for 60 minutes. Then, cells were incubated in a cocktail solution containing goat anti-CB1 antibody (1:500; Santa Cruz Biotechnology Inc.) and mouse anti-nestin antibody (1:2,000; Chemicon International) at 4°C overnight. The cells were washed in PBS 3 times and incubated in another cocktail solution containing Alexa Fluor 488–conjugated rabbit anti-mouse IgG (1:400) and Alexa Fluor 568-–conjugated rabbit anti-goat IgG (1:400) secondary antibodies (Invitrogen Corp.) at room temperature for 3 hours. Cell nuclei were stained in PBS containing 5 μg/ml of Hoechst 33258 (Sigma-Aldrich) at room temperature for 20 minutes. The coverslips were washed and mounted with antifade medium (DakoCytomation). Immunohistochemical controls for CB1 antibody were performed using antiserum preabsorbed with the immunogen (1.0 mg/ml).
For differentiation experiments, dissociated cells were plated on 12-mm glass coverslips coated with 15 μg/ml of poly-L-ornithine within 24-well culture plate at a density of 3 × 104 cells per well and were cultured in DMEM/F12 medium supplemented with B27 supplement, 20 ng/ml FGF-2, and 20 ng/ml EGF for 12 hours. Then, the culture medium was changed to FGF-2– and EGF-free DMEM/F12 medium containing B27 supplement and HU210 (10 or 100 nM or 1 μM) or AEA (1 or 5 μM). Cells were cultured further for 8 days, with complete change of culture medium and drugs once every 3 days. After fixation with 4% paraformaldehyde, cells were rinsed in PBS and incubated in 5% normal goat serum for 60 minutes before incubation in mouse anti-TuJ1 antibody (1:500; Sigma-Aldrich) at 4°C overnight. The primary antibody was visualized by incubation of cells in Alexa Fluor 568–conjugated goat anti-mouse secondary antibody (1:400; Invitrogen Corp.) at room temperature for 3 hours. Cell nuclei were stained with Hoechst 33258 solution for 20 minutes. The number of TuJ1-positive cells and Hoechst-stained total cells was counted using a 40× objective lens on an Olympus BX-51 light microscope. Cell counting was conducted in 10 randomly chosen fields in each coverslip by an observer who had no knowledge of the treatment conditions.
Western blotting.
NS/PCs were plated on 10-cm culture dishes coated with 15 μg/ml of poly-L-ornithine at 1 × 106 cells/dish for 24 hours. Then, 100 nM of HU210 or vehicle was added to the culture medium, and the cells were collected 0, 5, 15, 30, and 60 minutes later. Total cellular proteins were extracted with lysis buffer (1% Triton X-100, 10% glycerol, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitor mix). Equivalent amounts of extracted proteins (20 μg) were resolved on 10% SDS-PAGE and electroblotted at 4°C for 50 minutes to nitrocellulose membrane (Amersham Biosciences). After blocking the background staining with 5% (wt/vol) skim milk in PBS, the membranes were incubated in goat anti-CB1 antibody (1:500; Santa Cruz Biotechnology Inc.), rabbit anti-pAkt (1:1,000; Alpha Diagnostic International), mouse anti-pERK1/2 (1:1,000; Santa Cruz Biotechnology Inc.), and rabbit anti–total ERK1/2 (1:1,000; Cell Signaling Technology). Antibody against β-actin (1:1,000; Sigma-Aldrich) was used as an internal control for the concentration of protein loaded. Immunoreactive proteins were detected using HRP-conjugated secondary antibodies and an ECL kit (Amersham Biosciences) according to the manufacturer’s instructions. Quantification of the immunoblots was performed by densitometric analysis of chemiluminescence-exposed films, using Image-Pro Plus software (version 4.1; Carsen Group). Immunohistochemical controls were performed by using antiserum preabsorbed with the immunogen (1.0 mg/ml).
NS/PCs were collected, and rat CB1 full encoding region was amplified in NS/PCs by PCR with primers (sense: 5′-TGGATCCATGAAGTCGATCCTAGATGGCC-3′; antisense: 5′-CGAATTCTCACAGAGCCTCGGCGGACGTG-3′). The amplification reaction was carried out in a PerkinElmer GeneAmp (PCR System 9600) for 30 cycles. Each cycle consisted of denaturation for 40 seconds at 94°C, annealing for 40 seconds at 55°C, and an extension for 120 seconds at 72°C. A final extension step at 72°C for 5 minutes terminated the amplification. PCR product was analyzed by electrophoresis using a 1% agarose gel.
HU210, AM281, and kainic acid treatment in adult rats.
Both HU210 and AM281 were dissolved in pure DMSO, and aliquots were stored at –80°C with the final working concentration of 1 ml/kg body weight. Two doses of HU210 were used, 25 or 100 μg/kg, which produce either nondetectable or severe behavioral changes, respectively, including ataxic walking and deep sleep. Only 1 dose of AM281, 3 mg/kg, was employed, because this dose effectively antagonizes the behavioral effects produced by HU210 (8). Two injection protocols were utilized. To detect the acute effects of cannabinoid on cell proliferation in the hippocampus, rats received a single injection of vehicle, 25 or 100 μg/kg of HU210, or 3 mg/kg of AM281. To examine the chronic effects, rats received twice daily injections of vehicle, 25 or 100 μg/kg of HU210, or 3 mg/kg of AM281 for 10 days. Three rats received kainic acid injection (10 mg/kg, i.p.), and 1 hour after epileptic status, rats received sodium pentobarbital to stop behavioral seizures (31). One day after kainic acid injection, rats were killed for TUNEL and Fluoro-Jade B staining.
BrdU labeling in adult rats.
Male Long-Evans, Wistar, and Fischer 344 rats weighing 220–250 g received i.p. injections of 50 mg/kg of BrdU (Sigma-Aldrich). Three BrdU injection paradigms were used. To identify the possible expression of CB1 receptors in dividing cells in the hippocampal dentate gyrus, rats received a single dose of BrdU 2 hours after HU210 and were perfused 2 hours later. To examine cell proliferation in the hippocampus, rats received 2 doses of BrdU (2 hours after the last HU210) with a 2-hour interval and were killed 24 hours after the last BrdU injection. To investigate differentiation of newborn cells in the hippocampus, rats received 4 doses of BrdU (12 hours after the last HU210) with 12-hour intervals and were allowed to survive for 1 month after the last BrdU injection.
Immunohistochemistry for adult rats.
Under anesthesia with sodium pentobarbital (50 mg/kg, i.p.), rats were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed, post-fixed for 2 hours in the same fixative, and placed in 20% sucrose until they sank. Coronal sections (40-μm thickness) through the entire hippocampus were cut on a sliding microtome and stored in PBS. Immunohistochemical detection of single BrdU labeling was conducted with previously described protocols (16). Briefly, free-floating sections were pretreated in 50% formamide/2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate) at 65°C for 2 hours and were incubated in 2 M HCl at 37°C for 30 minutes. After a 10-minute wash in 0.1 M borate buffer (pH 8.5) to neutralize the HCl, sections were incubated in mouse anti-BrdU antibody (1:1,000; Sigma-Aldrich) at 4°C for 36 hours. Sections were washed again and incubated in biotinylated goat anti-mouse secondary antibody (1:250; Vector Laboratories) for 2 hours at room temperature, followed by wash in PBS and an incubation in avidin-biotin-peroxidase complex for 2 hours. After a final wash, sections were reacted for peroxidase enzyme activity by using 3,3′-diaminobenzidine. Immunohistochemical detection of single NeuN labeling using mouse anti-NeuN antibody (1:2,000; Chemicon International) was conducted with protocols similar to those described above, except pretreatment with formamide and HCl was omitted. The specificity of immunolabeling was verified by controls in which the primary antibody was omitted.
For double immunofluorescence staining, DNA in the sections was first denatured to expose the antigen, as described above. Sections were then incubated at 4°C for 36 hours in a cocktail solution containing sheep anti-BrdU antibody (1:500; Cedarlane Laboratories Ltd.) and mouse anti-NeuN antibody (1:500; Chemicon International). After multiple washes in PBS, sections were incubated for 4 hours in Alexa Fluor 488–conjugated donkey anti-sheep secondary antibody preabsorbed with goat IgG (1:400; Invitrogen Corp.) and Alexa Fluor 568–conjugated goat anti-mouse secondary antibody (1:400; Invitrogen Corp.). Sections were mounted on gelatinized slides and coverslipped.
TUNEL stain and Fluoro-Jade B stain.
Three groups of 3 rats each receiving no treatment (control), chronic HU210 injection, and kainic acid injection were perfused with 4% paraformaldehyde as described above. Several series of coronal sections (40-μm) through the hippocampus were cut on a sliding microtome. One series of sections was stained with Fluoro-Jade B stain according to the manufacturer’s instruction (Histo-Chem Inc.) and our previous study (31). Adjacent sections were stained with a TUNEL staining kit according to the manufacturer’s instruction (Trevigen Inc.).
Quantification of BrdU and NeuN labeling.
Every sixth section through the dorsal hippocampus was processed for BrdU immunohistochemistry, resulting in each section being 240 μm apart, thus ensuring the same cell would not be counted twice. All BrdU-positive cells in the entire granule cell layer (superior and inferior blades), SGZ (defined as a 2-cell-body-wide zone along the border of the granule cell layer), and the hilus were counted in each section by an experimenter who was not informed of the group assignment. The total number of BrdU-labeled cells per section was then averaged. This cell profile counting method is theoretically comparable to the true stereology cell counting, because we conducted random sampling (i.e., every sixth section was sampled), avoided double counting of the same cells (each section was 240 μm apart), and counted all the cells labeled with BrdU regardless of their shape and size or of the volume of the dentate gyrus. Two recent studies have used similar cell counting methods (59, 60), which showed results indistinguishable from those provided by stereology cell counting (61).
For quantification of BrdU/NeuN double-labeling cells, immunofluorescence images were obtained under a confocal laser-scanning microscope (LSM 510 META; Zeiss). Fifty BrdU-positive cells in each rat were randomly selected and then analyzed by orthogonal reconstructions from z-series (z-step, 1 μm) to obtain the proportion of BrdU/NeuN double-labeling cells to BrdU-stained cells.
Stereological cell counting was performed for quantification of the total number of NeuN-stained neurons in the hippocampus of 2 groups of 3 rats each receiving no treatment (control) or chronic HU210 injection. We used an Olympus BX-51 microscope interfaced with a color digital camera (MicroFire; Optronics) and analyzed using the image analysis computer software Stereo Investigator (version 4.10; MicroBrightField Inc.). The analysis of the sections started from a random position at the origin of the hippocampus. Every sixth section was included such that the rostral 7 sections through the dorsal hippocampus in each animal were analyzed, and these 7 sections cover over 90% of the dorsal hippocampus. The contour delineations (left and right) of the dentate granule cell layer and CA3 pyramidal cell layer medial to an artificial line connecting the tips of the 2 granule cell blades were drawn using a 2.5× lens. Cells were counted using the optical fractionator method (62). Each counting frame was placed at an intersection of the lines forming a virtual grid that was randomly generated and randomly placed by the software within the granule or pyramidal cell layer. The software also controlled the motorized stage of the microscope that allowed scanning of the entire brain region by successively meandering from one counting frame to the other. Cells were counted using a 100× oil lens (NA 1.40) and were included in the measurements only when they came into focus within the dissector (dissector height, 11 μm; average thickness of mounted sections was 13 μm). The software calculated the estimated total number of cells in each brain structure. The precision of the individual estimations is expressed by the coefficient of error. This value expresses the intra-individual variation due to the stereological estimating procedure (i.e., sampling of sections and counting locations), thus providing the information necessary for determining whether more or less sampling should be performed. The inter-animal variation in total cell number is given by the coefficient of variation. Counting was performed blind to the treatment, and the sections were decoded only after completion of the analysis.
Behavioral testing.
Rats received twice-daily injections of vehicle, AM281 (3 mg/kg), or HU210 (100 μg/kg) for 10 days, followed 12 hours later by 4 BrdU injections at 12 hour intervals. Rats were subjected to behavioral testing 1 month later. NSF testing was conducted according to a previously described protocol (63). Food was removed from the home cage 48 hours before testing, but water was provided. Prior to testing, a single pellet of food weighing 3.5–4.5 g was placed on a platform in the center of the testing plastic box (52 × 48 × 32 cm) with the floor covered with a thin layer of wooden bedding. Rats were placed individually in a corner of the box and a stopwatch immediately started. The measure of interest was the latency to begin eating, defined as chewing of the food, not simply sniffing or playing with it. If animals had not eaten within 600 seconds, the test was terminated and the animals were assigned a latency score of 600 seconds. Immediately after the test, rats were transferred to their home cages, and the latency to feed and the amount of food consumed in 15 minutes were measured, according to our pilot experiments. The whole process was videotaped and the latency to begin eating in the novel environment was measured by 2 experimenters blind to the treatment conditions.
For the FST, rats were placed individually in the testing plastic cylinder (54 cm high × 35 cm in diameter) containing a 38-cm water column (22 ± 1°C), according to previous studies (41, 42). Water was replaced between every trial. Two swimming sessions were conducted: an initial 15-minute pretest, followed by a 5-minute test 24 hours later. Following swim sessions, rats were removed from the cylinder, dried with a towel, and placed underneath a heating lamp for approximately 30 minutes before being returned to their home cages. Both pretest and test sessions were videotaped. Two observers blind to the treatment conditions scored the number of rats climbing in the first 5 minutes in the pretest session and for time spent immobile in the test session. Immobility was defined as floating passively in the water and only making slight movements to keep the head above the water.
Three groups of 5 rats each received twice-daily injections of vehicle (group 1), HU210 (group 2; 100 μg/kg), or HU210 (group 3; 100 μg/kg) for 10 days, followed 12 hours later by 4 BrdU injections at 12-hour intervals. All the rats were subjected to behavioral testing 1 month later. Group 3 rats also received irradiation using a Philips RT250 orthovoltage unit operated at 250 kV and 15 mA. Pre-irradiation dosimetry was performed using a PTW Unidos Electrometer type 10002 with Capintec Inc. Ionization Chamber model PR-06G. According to the method of Santarelli et al. (18), anesthetized rats were protected with lead shields that covered the entire body except the hippocampus. Dose rate measurements were performed with the ionization chamber (including its buildup cap) placed on the Perspex sheet. Readings were corrected for air temperature and pressure and indicated a dose rate of 67.2 cGy/min at a source-to-surface distance of 45 cm. The total dose was 5 Gy. Two 5 Gy doses were delivered 7 days apart, and the first dose was given 1 day after the first HU210 injection.
All results are expressed as mean ± SEM. Statistical analysis of the data was performed using standard 1-way ANOVA or 1-way ANOVA for repeated measures, followed by the Tukey post-hoc test. A 2-tailed Student’s paired t test was also used to compare the difference in values between 2 groups. Statistical significance was set at P < 0.05.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Saskatchewan to X. Zhang, who is the recipient of the CIHR New Investigator Award. W. Jiang and S.-P. Ji were supported by Postdoctoral Fellowship Award from the Saskatchewan Health Research Foundation. We thank Y. Li and G. Kort for technical assistance.

Yun Zhang and Lan Xiao contributed equally to this work.
Nonstandard abbreviations used: AEA, anandamide; FST, forced swimming test; NeuN, neuronal nuclear antigen; NSF, novelty-suppressed feeding; NS/PC, neural stem/progenitor cell; pERK1/2, phospho-ERK1/2; SGZ, subgranular zone of the dentate gyrus; TuJ1, β-tubulin III.

Conjugated linoleic acid improves blood pressure by increasing adiponectin and endothelial nitric oxide synthase activity

So I wonder if this is really telling me that CLA creates NO - nitric oxide. I would like that rather than having to take L-Carnitine and L-Arginine

Conjugated linoleic acid (CLA) has been reported to reduce blood pressure in obese insulin-resistant rats, but its mechanism of action has not been identified. The objective of this study was to determine whether CLA isomers can reduce obesity-related hypertension in the fa/fa Zucker rat in relation to adiponectin production and endothelial nitric oxide synthase (eNOS) activation. Obese fa/fa Zucker rats were randomly assigned to one of four groups: (1) cis-9,trans-11-CLA, (2) trans-10,cis-12 (t10,c12)-CLA, (3) control or (4) captopril. After 8 weeks, systolic blood pressure increased 30% in control obese rats. This increase was attenuated 11%–13% in the t10,c12-CLA isomer and captopril groups, respectively. The t10,c12-CLA isomer concurrently elevated adiponectin levels in both plasma and adipose tissue and increased phosphorylated eNOS in adipose tissue as well as the aorta. Although a direct effect of CLA was not observed in cultured endothelial cells, direct adiponectin treatment increased phosphorylation of eNOS. Endothelial nitric oxide synthase phosphorylation was also increased in adipose of fa/fa Zucker rats infused with adiponectin in parallel with improvements in blood pressure. Our results suggest that the t10,c12-CLA isomer attenuates development of obesity-related hypertension, at least in part, by stimulating adiponectin production, which subsequently activates vascular eNOS.

ARISTOTLE: Apixaban noninferior to warfarin in AF patients

Anything has to be better than rat poison and all the blood tests needed.
Princeton, NJ and New York, NY - Topline results from the ARISTOTLE trial, comparing apixaban (Eliquis, Pfizer/Bristol-Myers Squibb [BMS]) to warfarin in subjects with atrial fibrillation [AF] and risk factors for stroke, suggest that the oral direct factor Xa inhibitor is noninferior to the older standard for the prevention of stroke and systemic embolism [1].

According to preliminary results of the study, released late Wednesday, apixaban also "met the key secondary endpoints of superiority on efficacy and on ISTH (International Society on Thrombosis and Haemostasis) major bleeding compared to warfarin."

Full results of the trial will be presented August 28 at the European Society of Cardiology 2011 meeting in Paris, France.

ARISTOTLE enrolled over 18 000 AF patients in over 1000 centers in roughly 40 countries, the press release notes. The trial randomized patients to either a twice-daily dose of apixaban 2.5 mg, or dose-adjusted warfarin.

If ultimately approved, apixaban would compete in this indication against dabigatran (Pradaxa, Boehringer Ingelheim), which is already on the US and other markets, as well as rivaroxaban (Xarelto, Bayer/Johnson & Johnson), still waiting for US approval. Rivaroxaban demonstrated noninferiority to warfarin in the ROCKET AF trial.

Earlier this year, apixaban proved itself superior to aspirin in the 5599-patient AVERROES study, conducted in patients with AF, at risk for stroke, who were not suitable candidates for warfarin therapy as reported by heartwire. Preliminary AVERROES results were also released early, after a predefined interim analysis by the independent data monitoring committee saw a clear and "clinically important reduction in stroke and systemic embolism."

The first approval for apixaban was last month, in Europe, where regulators granted it approval for use in the 27 countries of the EU for the prevention of venous-thromboembolic (VTE) events in adult patients who have undergone elective hip- or knee-replacement surgery.

High-dose statin therapy increases the risk of diabetes: Meta-analysis

You can add this to the other risks of statins.
London, UK - A meta-analysis of some of the more high-profile statin trials testing the effectiveness of high-dose therapy has revealed a significant increase in the risk of diabetes mellitus associated with statin use in high doses [1]. Compared with moderate-dose therapy across five statin trials, investigators report that treatment with high-dose statins increased the risk of diabetes by 12%.

Senior investigator Dr Kausik Ray (St George's University of London, UK) said that while there might be consequences from the raised blood glucose levels, researchers do not yet know what these long-term effects mean. The net benefit of high-dose statin therapy "is definitely in favor" of using the drugs, he said.

"One thing we do know is that there does appear to be a dose effect with statin therapy, with the risk of diabetes mellitus increasing with higher doses," Ray told heartwire. "Statins have multiple effects and cause a number of changes. What we're seeing is probably an off-target effect, and right now we have no obvious mechanisms. However, lowering LDL-cholesterol levels is probably more important than the increase in blood-sugar levels."

In their analysis, the number of patients needed to treat with high-dose statin therapy to prevent one cardiovascular event was 155, whereas the number needed to treat to cause one case of new-onset diabetes mellitus was 498. Overall, high-dose statin therapy reduced the risk of cardiovascular events in their meta-analysis by 16% compared with low- or moderate-dose statin therapy.

The results of the study are published in the June 22, 2011 issue of the Journal of the American Medical Association.

Previous observed diabetes risk

Speaking with heartwire, Ray said that the idea for the meta-analysis began two years ago, when the signal for diabetes risk was observed in Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER). The group later performed an analysis of some of the early statin trials comparing the lipid-lowering drugs with placebo in 90 000 individuals and observed a significant 9% increase in the risk of diabetes mellitus. That study was reported in the Lancet in 2010 [2].

In this newest analysis, the researchers included large, randomized, controlled, hard-end-point studies that compared intensive-dose statin therapy with moderate-dose statin therapy followed for more than one year. The trials included in the meta-analysis were PROVE-IT, A to Z, TNT, IDEAL, and SEARCH, five trials that together included 32 752 patients without diabetes mellitus at baseline.

"We wanted to look at the different studies comparing the intensity of statin treatment," said Ray. "If the diabetes finding was a real finding, we would expect to see it in the statin trials that tested different intensities of treatment in about 33 000 subjects. The trials all ranged from two to five years in duration, and we had information from five trials comparing high versus low/moderate treatment doses. Our results support our initial findings. We observed a 12% increase in risk for those patients treated with high-dose statin therapy."

In total, 1449 patients treated with high-dose statin therapy developed diabetes compared with 1300 patients assigned to moderate-dose statin therapy. This translated into two additional cases of diabetes mellitus per 1000 patient-years. The odds ratio for new-onset diabetes was 1.12 (95% CI 1.04-1.22). Regarding benefit, 3134 patients treated with high-dose statin therapy and 3550 patients treated with moderate-dose therapy had a cardiovascular event, translating into 6.5 fewer outcomes per 1000 patient-years in the high-dose statin arm, or a relative reduction of 16%.

The investigators did observe differential effects with the different drugs. Whereas atorvastatin 80 mg and simvastatin 80 mg were both associated with similar risks of diabetes mellitus, the benefit differed significantly, with evidence in favor of atorvastatin (22% vs 5% risk reduction for cardiovascular events). The data, said Ray, support the recent Food and Drug Administration (FDA) decision to warn physicians to not start new patients on simvastatin 80 mg and to be vigilant to the risks of muscle toxicity caused by the drug in those who are still taking it.

Getting patients to goal

Speaking with heartwire about the results, Dr Brendan Everett (Brigham and Women's Hospital, Boston, MA), who was not involved in the analysis, agreed with the conclusions of Ray and colleagues, that the signal observed in this latest analysis supports the findings from JUPITER and the Lancet meta-analysis "and supports the idea of a dose effect, that there is an increasing risk of diabetes with increasing doses of statins."

Everett said the investigators helped clinicians by providing data on the relative benefits and relative harms of high-dose statin therapy. The number needed to treat to prevent one cardiovascular event and number needed to treat to cause one new case of diabetes mellitus clearly support the use of high-dose statin therapy in the patients studied in the five clinical trials.

"The benefits of statins for reducing important macrovascular events is so overwhelming that the balance is clearly on the side of benefit," said Everett. "This is an important point that shouldn't be forgotten."

Dr Roger Blumenthal (Johns Hopkins University Medical Center, Baltimore, MD) agreed, stating that while "it makes sense that higher doses [of statins] would have slightly higher adverse effects," there is still no proposed mechanism for the increased risk of diabetes. Like the others, he told heartwire that the benefits of moderate/high doses of statins outweigh the risks, although he added that some physicians might decide to downgrade the dose based on these new data.

In addition, Dr Steven Nissen (Cleveland Clinic, OH) told heartwire that the effect is likely real as it has been observed in enough trials and analyses. That said, "it is notable that despite the increase in the risk of diabetes mellitus, the reduction in cardiovascular morbidity and mortality is maintained," he added. "It leads me to believe that the effect is not very clinically significant."

Everett added that what is currently unknown is how the risks of diabetes mellitus differ in other patient populations. Clinicians need to understand their patients' baseline risks of cardiovascular disease and diabetes mellitus when making a decision about high-dose statin therapy, and it will be important to determine whether patients at greater risk for adverse side effects can be identified. Researchers will also need to determine what effects high-dose statin therapy has on microvascular complications, such as retinopathy.

"The bottom line is that we need to follow up on the signal, but I don't believe the results should change treatment goals," said Everett. A failure to aggressively treat patients at high risk for cardiovascular events will result in an excess of clinical events, he added.

To heartwire, Ray suggested that clinicians monitor HbA1c levels when treating patients with high-dose statin therapy.

Like Ray, Everett said the results support the FDA decision regarding simvastatin 80 mg, that the drug is associated with more side effects without a corresponding balance of efficacy. In the interest of getting patients to treatment goal and trying to do so in a cost-efficient manner, some physicians had been using high-dose simvastatin. However, if they are unable to get to goal at 40-mg simvastatin, switching over to other, nongeneric drugs is not difficult, although it does involve extra paperwork when dealing with drug payers, he said.

This Sunday at the American Diabetes Association (ADA) 2011 Scientific Sessions, Dr David Preiss (University of Glasgow, Scotland), the first author of the analysis, will present their data at a special symposium organized by the ADA and FDA. The presentation will highlight the newly observed risks with high-dose statin therapy, as well as their Lancet analysis of 13 randomized trials comparing placebo and standard-therapy trials.