Reduced Post-ischemic Brain Injury in Transient Receptor Potential Vanilloid 4 Knockout Mice

A 'promising target' which will never occur because we have NO STROKE LEADERSHIP AND NO STROKE STRATEGY. Aren't you just fucking glad that such cesspools of incompetence means your children and grandchildren will suffer just as much from strokes as you are?

Reduced Post-ischemic Brain Injury in Transient Receptor Potential Vanilloid 4 Knockout Mice

Koji Tanaka¹, Shoji Matsumoto², Takeshi Yamada³, Ryo Yamasaki¹, Makoto Suzuki⁴, Mizuho A. Kido⁵ and Jun-Ichi Kira^1*

• ¹Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
• ²Department of Comprehensive Strokology, School of Medicine, Fujita Health University, Toyoake, Japan
• ³Department of Neurology, Saiseikai Fukuoka General Hospital, Fukuoka, Japan
• ⁴Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical University, Shimotsuke, Japan
• ⁵Department of Anatomy and Physiology, Faculty of Medicine, Saga University, Saga, Japan

Background and Purpose: 

In the acute phase of ischemia-reperfusion, hypoperfusion associated with ischemia and reperfusion in microvascular regions and disruption of the blood–brain barrier (BBB) contribute to post-ischemic brain injury. We aimed to clarify whether brain injury following transient middle cerebral artery occlusion (tMCAO) is ameliorated in Transient receptor potential vanilloid 4 knockout (Trpv4^–/–) mice.

Methods: 

tMCAO was induced in wild-type (WT) and Trpv4^–/– mice aged 8–10 weeks. Ischemia-induced lesion volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining at 24 h post-tMCAO. Tissue water content and Evans blue leakage in the ipsilateral hemisphere and a neurological score were evaluated at 48 h post-tMCAO. Transmission electron microscopy (TEM) was performed to assess the morphological changes in microvasculature in the ischemic lesions at 6 h post-tMCAO.

Results: 

Compared with WT mice, Trpv4^–/– mice showed reduced ischemia-induced lesion volume and reduced water content and Evans blue leakage in the ipsilateral hemisphere alongside milder neurological symptoms. The loss of zonula occludens-1 and occludin proteins in the ipsilateral hemisphere was attenuated in Trpv4^–/– mice. TEM revealed that parenchymal microvessels in the ischemic lesion were compressed and narrowed by the swollen endfeet of astrocytes in WT mice, but these effects were markedly ameliorated in Trpv4^–/– mice.

Conclusion: 

The present results demonstrate that TRPV4 contributes to post-ischemic brain injury. The preserved microcirculation and BBB function shortly after reperfusion are the key neuroprotective roles of TRPV4 inhibition, which represents a promising target for the treatment of acute ischemic stroke.

Introduction

Acute ischemic stroke secondary to large artery occlusion is a common and devastating condition that results in death or long-term disability in a high proportion of patients. Recent advances in revascularization therapies, including mechanical thrombectomy, have enabled rapid and effective recanalization for large artery occlusions in patients with acute ischemic stroke (Goyal et al., 2016). However, despite the high rates of early recanalization, some patients still experience poor outcomes. In these cases, lack of reperfusion due to blockade of the microcirculation leading to hypoperfusion associated with ischemia and reperfusion and disruption of the blood–brain barrier (BBB) may contribute to post-ischemic brain injury (Molina and Alvarez-Sabín, 2009).

Transient receptor potential vanilloid 4 (TRPV4), a member of the TRP vanilloid subfamily, is a non-selective cation channel permeable to Ca²⁺, Na⁺, and Mg²⁺ ions (Ramsey et al., 2006). TRPV4 was first described as a cellular osmotic sensor that detects extracellular hypo-osmolarity, and was subsequently shown to be activated by multiple stimuli. TRPV4 is widely expressed in the central nervous system (Nilius and Voets, 2013), and its localization on astrocyte endfeet plays an essential role in the regulation of vascular tone in brain microvessels (Dunn et al., 2013; Filosa et al., 2013). Enrichment of TRPV4 and aquaporins at the endfeet of astrocytes ensures that even small changes in conditions, such as extracellular ion concentrations, result in astroglial swelling, volume regulation, and reorganization of downstream signaling pathways (Ryskamp et al., 2015; Iuso and Križaj, 2016). Pharmacological blockade of TRPV4 results in reduction of brain ischemia-induced lesion volume through inhibition of matrix metalloprotease (MMP) activation, thereby providing protection against BBB disruption following transient focal cerebral ischemia (Li et al., 2013; Jie et al., 2015a, b, 2016). Moreover, TRPV4 contributes to Ca²⁺ influx in both astrocytes and neurons and to extracellular glutamate accumulation during peri-infarct depolarizations (Rakers et al., 2017). These findings indicate that TRPV4 has a large influence on ischemia-induced brain injury and indicate that inhibition of TRPV4 has a neuroprotective role in acute ischemic stroke. However, a recent report demonstrated exacerbation of ischemia-induced brain edema formation in Trpv4 knockout (Trpv4^–/–) mice (Pivonkova et al., 2018). Specifically, Trpv4^–/– mice showed larger T2-hyperintensity lesions following transcranial permanent middle cerebral artery occlusion (pMCAO) compared with wild-type (WT) mice. Moreover, the report described a minimal involvement of TRPV4 in astrocyte volume regulation in vivo. Thus, the mechanism for the neuroprotective effects of TRPV4 in acute ischemic stroke remains inconclusive. Given this context, we aimed to investigate the differences in post-ischemic brain injury between Trpv4^–/– and WT mice, and to clarify the mechanism for any identified differences using a transient MCAO (tMCAO) model in the C57BL/6N mouse strain.

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