Correlation between Arteriole Membrane Potential and Cerebral Vasospasm after Subarachnoid Hemorrhage in Rats
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.280652
Source of Support: None, Conflict of Interest: None
Keywords: Arteriole, cerebral vasospasm, membrane potential, subarachnoid hemorrhageKey Messages: The Em values of arterioles depolarize after SAH, and a significant correlation is found between the Em of arterioles and diameter of basilar artery.
Aneurismal subarachnoid hemorrhage (SAH) accounts for 5% of all strokes and delayed vasospasm and develops in approximately 70% of patients between 3 and 14 days after SAH., Research showed that delayed ischemic neurological deficits or delayed cerebral ischemia (DCI), which is caused by cerebral artery vasospasm after SAH, leads to ultimate infarction and poor outcome. Cerebral vasospasm (CVS) usually occurs on day three after SAH, peaks at days 6 and 8, and lasts for 2–3 weeks in patients, and it was shown to induce vasospasm in cerebral artery in experimental studies. Furthermore, there are a number of animal studies that are consistent with the clinical studies that reported that reducing angiographic vasospasm improves outcome. However, double-blind experiments and reversing vasoconstriction do not improve patient outcomes., Thus, other mechanisms are presented, including early brain injury, cortical spreading depression, microcirculatory dysfunction, and microthrombosis.,,,
In microcirculatory dysfunction, microthrombosis and microarterial constriction are mainly studied., Microvessel constriction after SAH was demonstrated in the animal model and clinical operation.,,, Substances derived from subarachnoid clots and substances that act on the inside wall of arterioles can cause vasoconstriction., Functional disturbance of arterioles can contribute to microthrombus formation. Arteriolar constriction and microthrombi are speculated to play an important role in DCI after SAH. Therefore, understanding the mechanisms of arteriole constriction and inhibition of constriction in arterioles could improve the outcomes of aneurismal SAH.
Vasospasm is a problem of smooth muscle contraction that involves alterations in membrane potential (Em);, therefore, electrophysiological studies have been performed on vascular smooth muscle (VSM) following experimental SAH.
The resting Em of VSM of artery recorded in vitro from the animal models of SAH was significantly depolarized.,,, However, the Em alteration of arterioles after SAH remained unclear and needed further investigation. This study examined the time-related Em alteration of arterioles isolated from SAH model rats.
Sprague–Dawley rats (either male or female, 300–350 g) were purchased from the Animal Center of Xinjiang Medical University, Xinjiang, China.
All experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Medical College, Shihezi University. Sprague–Dawley rats were randomly assigned into control (n = 30), sham (n = 30), and SAH (n = 30) groups. In contrast to the sham and the SAH groups, the control group was treated using the same protocol as described below, except that no blood or saline was injected into the subarachnoid space. The sham group was injected with equal volumes of saline (100 μL, 0.9% NaCl) in the prechiasmatic cistern, and the SAH model was induced by injecting fresh and non-heparinized arterial blood into the prechiasmatic cistern.
Prechiasmatic cistern subarachnoid hemorrhage model
In brief, the rats were anesthetized with intraperitoneal injections of chloral hydrate (350 mg/kg), and their heads were fixed in a stereotactic frame. At the right side of the skull, a hole was drilled through the skull bone down to dura mater without perforation. The hole site was 5 mm anterior to the bregma and 4 mm lateral from the midline. A PE 10 cannula was tilted 45° in the sagittal plane and was introduced to approximately 10 mm from the bone hole. When the cerebralspinal fluid (CSF) outflowed from the cannula, 0.2 mL of non-heparinized blood was withdrawn from the femoral artery and injected intracranially through the cannula into prechiasmatic cistern in 20 s. Finally, the hole was plugged by bone wax. The second blood injection performed after 48 h.
Basilar artery and arteriole collection
According to the time points of the sacrificed animals, each group was divided into six subgroups, including 1, 3, 5, 7, and 14-day groups after the second operation.
Rats were sacrificed under deep anesthesia, and the brains were collected to confirm the success of the building model. Basal cisterns and other cisterns were stained with blood, and red blood cells were found in the subarachnoid space by using hematoxylin and eosin (HE) staining [Figure 1]. After deep anesthesia, the chest was opened and a needle was inserted into the left ventricle, and cold saline was infused to wash blood. The brain was immediately placed in physiologic salt solution (PPS) composed of 118.9 mM NaCl, 4.69 mM of KCl, 1.17 mM MgSO4·7H2O, 1.18 mM KH2 PO4, 2.5 mM of CaCl2, 25 mM NaHCO3, 0.026 mM of ethylene diamine tetracetic acid, and 8.3 mM glucose with 95% O2, and 5% CO2. A segment of the basilar artery (2–3 mm in length) between the anterior inferior cerebellar arteries and the basilar artery was harvested to measure the diameter by using a pressure myograph.
After harvesting the basilar artery segments, the remaining arteries and brain tissue were transferred under a microscope at a magnification of 10 × 20 or 10 × 40. Arteriolar segments (40–80 μm in diameter, 0.4 mm in length) were harvested from the branches of the anterior inferior cerebellar artery in the pia. For whole cell recordings of SMCs of the arteriolar segments, arteriolar segments were placed in culture dishes (35 mm in diameter). Both ends of the arterioles were secured by small platinum pieces, and the arterioles were incubated 30 min longer in the perfusate with collagenase A. The perfusate was composed of 138 mM NaCl, 5 mM KCl, 1.6 mM CaCl2, 1.2 mM MgCl2, 5 mM Na-HEPES, 6 mM HEPES, and 7.5 mM glucose. After completely washing out the collagenase A with perfusate, the arterioles were further cleaned of its adventitial tissue until no connective tissue was found in the outer layer of the smooth muscle cells [Figure 2].
Tight-seal whole cell recordings
Whole-cell clamp recordings were performed on the arterioles. Membrane currents were acquired with an Axopatch 700 B amplifier (Sutter, USA) and filtered at 2 or 5 kHz. Electrodes were pulled from the glass tubes (Sutter, USA) with P-97 control instrument (Sutter, USA). The internal solution in the electrodes contained 130 mM gluconate, 10 mM NaCl, 2.0 mM CaCl2,1.2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, and 7.5 mM glucose (pH 7.4 and an osmolarity of 290 mosM). The cells were sealed in patch-clamp micromanipulators with a seal resistance of 1–2 GΩ. Whole-cell clamp conditions were obtained by applying suction or electric stimulation to the micromanipulators to break the membrane. Pipette capacitance was well compensated after a gigaseal with the cell was achieved, and membrane filtered at 1–5 kHz currents were acquired. Bioelectric and signal data of Em was observed and recorded at sampling intervals of 10, 20, or 100 μs. Bioelectricity signal was recorded on a computer from a digital analog converter, and Axoscope 10.2 software (Axon Instruments, USA) was used for recording and analysis.,
Measure the diameter of basilar artery
The isolated basilar artery (2–3 mm in length) was transferred into a pressure myograph chamber of Myoview (DMT, Denmark) with PPS solution, which was completed within 45 min from the death of the rats. Subsequently, the vessel was sleeved on a glass cannula with 11-0 suture and continuously superfused with 95% O2 and 5% CO2 PPS at 37°C. The pressure in the vessel lumen increased from 0 mmHg to 10 mmHg every 5 min until the vessel developed spontaneous diastolic activity. The vessels that developed substantial spontaneous constriction were used to measure the diameter. When the pressure was set to 70 mmHg, diastolic activity was found in the vessel, and the diameter of the basilar artery was measured using Diamtrak software.,
The data were presented as the mean ± SEM and analyzed using Statistical Package for the Social Sciences (SPSS, Version 13.0, IBM Corporation, NY, USA). For multiple groups, data were analyzed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. The correlation between the diameter of the basilar artery and Em value was evaluated using Pearson correlation coefficients.
No animals died in the control and sham groups. In the SAH group, 13 out of 30 animals died, with a mortality rate of 43.33%. To conduct the experiment, we added the 13 rats subjected to SAH models to the SAH group.
[Figure 3] shows the resting Em recorded in mV in all groups. Statistical difference in Em values was not observed between the control and sham group. However, the Em of arterioles in the SAH group was depolarized, and the Em values (−22.43 ± 2.5 mV, −12.22 ± 2.9 mV, and − 3.6 ± 0.23 mV) were elevated in the SAH group on days 3, 5, and 7. The Em values in the SAH group were significantly higher than those in the control group (P < 0.01) and sham (P < 0.05 or P < 0.01) groups on day 3, 5, and 7. The Em values of − 32.22 ± 3.4 mV, −32.97 ± 2.0 mV, and − 30.5 ± 3.5 mV were elevated in the SAH group on days 3, 5, and 7, respectively.
[Figure 4] shows no statistical difference in the diameter between the control and sham groups. The basilar artery diameters were 297.37 ± 2.14 μm, 282.29 ± 2.23 μm, 264.91 ± 1.65 μm, 244.25 ± 2.75 μm, and 289.79 ± 1.71 μm in the SAH group on days 1, 3, 5, 7, and 14, respectively. In the SAH group, the diameters significantly decreased compared with those in control (P < 0.05 or P < 0.01) and sham (P < 0. 01) groups at all time points.
Pearson correlation coefficients were used to determine the relationship between the Em and diameter [Figure 5], and a significant correlation was observed (r = −0.8783, P < 0.01).
The Em values of arteriole and the diameter of basilar artery alteration at five different time points were observed within 14 days in SAH animals. This study shows that the Em values of arterioles increased on day 3, 5, and 7, and the diameter of the basilar artery decreased on day 1, 3, 5, 7, and 14. A significant correlation was found between the Em of arterioles and diameter of basilar artery. The experiment confirms that the Em values of arterioles depolarize after SAH. This result indicates that arterioles can constrict in response to spasmogenic substances derived from subarachnoid clots.
Arteriolar constriction has an important role in DCI; thus, its mechanism is studied in recent years. In arteriole constriction, the substances derived from clots in the subarachnoid space were reported to act on pial and parenchymal arterioles by infiltrating into the perivascular space. These substances also affect the arteriole wall from the inside of the vascular space. Large cerebral arteries release vasospastic substances during vasospasm, and these substances can induce constriction in peripheral arterioles. Finally, changes of ion flow lead to smooth muscle cell contraction. Potassium channels are the major determinants of resting membrane voltage, and hence, the regulators of vascular tone. Membrane depolarization increases the open probability of voltage-sensitive Ca 2+ channels and results in Ca 2+ influx and myocytes contraction.
Herz found pial microvessels constriction in guinea pig SAH model. Hart demonstrated a decreased external diameter of arteriole and an increased wall thickness in cat SAH model. Ohkuma revealed the change in internal diameter and wall thickness in 3 and 7 days in parenchymal arterioles and perforating arteries after SAH. In clinics, microvessel constriction is directly observed by operative microscope and indirectly confirmed by orthogonal polarization spectral imaging. The abovementioned research work mainly aimed at morphometric examination. In this study, arterioles constriction was demonstrated by electrophysiological monitoring in the rat SAH model. To meet the required patch clamp technique, we collected arterioles from the perforating arteries of the Anterior Inferior Cerebellar Artery (AICA), making the removal of connective tissue around the arterioles easy. The results of our study confirm that perforating arterioles, not parenchymal arterioles, constrict after SAH. Early research has identified parenchymal arterioles constriction. Increasing number of studies indicate constriction of pial arterioles and perforating arterioles.
In this study, the Em of arterioles depolarized on day 3, elevated maximally (−3.26 ± 0.2 mV) on day 7, and then returned to normal on day 14. Waters et al. measured the Em of the basilar artery SMCs by glass microelectrodes with tip sizes less than 0.1 μm in the cat SAH model. Em was measured at 14 time points between 30 min and 7 days after injecting blood into the cistern. Similar to the result of this study, the Em of SMCs in the basilar artery depolarized. However, differences existed between the two experiments. The Em of SMCs depolarized as early as 30 min, and the depolarization state persisted until day 7. Although the data of Em were not analyzed in the SAH group, the peak levels of Em appeared at 12 h (−42.3 ± 1.9 mV) and 21 h (−42.8 ± 3.4 mV). Reasons for the differences in results are as follow. First, the animal models in our experiment were different. Second, in our experiment, blood was injected into the prechiasmatic cistern, which is located at the anterior circulation, whereas in the other experiment blood was injected into the cistern magna, which is located at the posterior circulation. Third, the test used different samples. Our study used arterioles from AICA, whereas the other study used SMCs of basilar artery. Clinical reports have shown that angiographic vasospasm is not always consistent with DCI. The difference in Em represents the difference of constriction between the arteriole and artery, which may explain the clinical phenomenon.
We also investigated the change of Em and diameter at various time intervals. Arteriole and basilar artery vasospasm were observed after SAH. However, their time phase change regulation was different. The basilar artery constricted at all time points (days 1, 3, 5, 7, and 14), whereas the arteriole constricted only on day 3, 5, and 7. This result indicates that the mean basilar artery vasospasm occurred early and lasted for a long time. Arteriole maximal luminal narrowing between days 3 and 7 was reported in vivo in mice subjected to other experimental SAH., Combined with the mechanism of microvessel spasm, we consider that the reason for this time difference was related to these factors. In this study, the wall of the cerebral artery was not damaged because the animal model was established through blood injection, therefore, the endothelial cells in the artery were not damaged at an early stage. The arterial wall stimulated by blood clot and its metabolites within the subarachnoid space, which had no vasospastic substances derived from the endothelial cells, induced spasms at an early stage. At this stage, substance-induced vasospasm had no contact with arterioles. Over time, bloody cerebrospinal fluid diffused into space around the arterioles. Meanwhile, endothelial cell damage caused by artery vasospasm may reduce the nitric oxide, and the activated platelets released vasospastic substances, which can induce the distal arteriole spasm through blood circulation. Finally, although arterioles dilate to maintain the cerebral blood flow after vessel spasm, there is a limit to such ability of compensation. With the decompensation of autoregulation, vessel narrowing may lead to significant drop in blood flow. Therefore, arteriole spasms occurred later than the large artery, which was similar to the previous results reported by Ohkuma. The arteriole returned to normal earlier than the artery, and we speculated that this may be related to the autoregulatory role of the brain. Although cerebral autoregulation was impaired after SAH, maintaining the cerebral blood flow is an inherent ability of blood vessels. Harper's dual-control hypothesis proposed that proximal arterial spasm may be compensated by distal autoregulatory vasodilatation. Arterioles are more prone to effect caused by the hypothesis with the thin wall.
The experiment confirms that the Em values of arterioles depolarized after SAH, which indicates that the arterioles can constrict in response to spasmogenic substances derived from subarachnoid clots. Similar to the artery, arteriole constriction is also involved in the pathophysiological events of delayed cerebral ischemia.
We thank Ketao Ma for help. This work was supported by the National Natural Science Foundation of China (No.81960222), the International Science and Technology Cooperation project of Shihezi University (No. GJHZ201704), and Doctoral Fund of the First Affiliated Hospital, School of Medicine, Shihezi University (No.BS201701).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]