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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: J Neurochem. 2021 Sep 20;159(5):887–900. doi: 10.1111/jnc.15496

Spontaneous, transient adenosine release is not enhanced in the CA1 region of hippocampus during severe ischemia models

Mallikarjunarao Ganesana 1, B Jill Venton 1,*
PMCID: PMC8627433  NIHMSID: NIHMS1734754  PMID: 34453336

Abstract

Ischemic stroke causes damage in the brain and a slow buildup of adenosine is neuroprotective during ischemic injury. Spontaneous, transient adenosine signaling, lasting only 3 seconds per event, has been discovered that increases in frequency in the caudate-putamen during early stages of mild ischemia-reperfusion injury. However, spontaneous adenosine changes have not been studied in the hippocampus during ischemia, an area highly susceptible to stroke. Here, we investigated changes of spontaneous, transient adenosine in the CA1 region of rat hippocampus during three different models of varied intensity of ischemia. During early stages of the milder bilateral common carotid artery occlusion (BCCAO) model, there were fewer spontaneous, transient adenosine, but no change in the concentration of individual events. In contrast, during the moderate 2 vertebral artery occlusion (2VAO) and severe 4 vessel occlusion (4VO) models, both the frequency of spontaneous, transient adenosine and the average event adenosine concentration decreased. Blood flow measurements validate that the ischemia models decreased blood flow and corresponding pathological changes were observed by transmission electron microscopy (TEM). 4VO occlusion showed the most severe damage in histology and BCCAO showed the least. Overall, our data suggest that there is no enhanced spontaneous adenosine release in the hippocampus during moderate and severe ischemia, which could be due to depletion of the rapidly releasable adenosine pool. Thus, during ischemic stroke, there are fewer spontaneous adenosine events that could inhibit neurotransmission, which might lead to more damage and less neuroprotection in the hippocampus CA1 region.

Graphical abstract.

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We investigated early changes in spontaneous adenosine release in the hippocampus in three different stroke models. We found that average event adenosine concentration decreases, while the inter-event time increases with increase in stroke intensity. These findings conclude that there is no enhanced adenosine release to act as a neuroprotectant during severe stroke.

Introduction

Adenosine is an endogenous neuromodulator and its concentration increases in the extracellular space after ischemia, causing neuroprotective effects (Melani et al., 2012). Administration of exogenous adenosine showed protection against CA1 hippocampal cell death and supported functional recovery following cerebral ischemic injury (Seydyousefi et al., 2019). While basal adenosine levels in the brain are in the nanomolar range (Melani et al., 2012), during ischemia, extracellular adenosine levels increased several fold (Melani et al., 2014). However, most studies measuring adenosine during ischemia were conducted on a timescale of minutes to hours, and fast changes cannot be observed (Pedata et al., 2005, 2007). Using fast-scan cyclic voltammetry (Ganesana et al., 2017; Shin et al., 2019; Xu et al., 2019), changes in spontaneous, transient adenosine events have been discovered that last only a few seconds in vivo (Cechova and Venton, 2008; Nguyen et al., 2014; Wang and Venton, 2019a) and in brain slices (Nguyen et al., 2015; Lee and Venton, 2018). In the caudate putamen, the frequency of these random events increased during bilateral common carotid artery occlusion (BCCAO) and thus the cumulative adenosine concentration increased by more than 50% during 2 hours of ischemia and reperfusion (Ganesana and Venton, 2018). Spontaneous, transient adenosine events were correlated with transient changes in oxygen, suggesting spontaneous, transient adenosine functions as a local neuromodulator, increasing local blood flow in response to ischemia and reperfusion (Wang and Venton, 2019b). However, changes in adenosine events have not been measured in other brain regions and also during more severe models of stroke.

Ischemia is a condition where blood flow to the brain is inhibited, and it is modeled by blocking, either transiently or permanently, blood vessels that provide blood flow to the brain (Jun Chen, Zao C. Xu, Xiao-Ming Xu, 2009). The carotid arteries are on the front of the neck and are one of the main source of blood flow to the hippocampus (Bhattacharjee et al., 2012). BCCAO can be performed transiently and reversibly by using balloon occluders, and it causes a milder ischemic insult because there is still collateral blood flow through the vertebral arteries (Coyle and Panzenbeck, 1990). Acute BCCAO results in oxidative stress leading to the disruption of the cell membrane and shrinkage of the cell nucleus (Gaur et al., 2009; Wahul et al., 2018; Serra et al., 2019). Two vertebral arteries occlusion (2VAO) is another model, in which both the vertebral arteries on the back of the neck are permanently cauterized. While the carotid arteries still provide collateral blood flow, permanent 2VAO causes significant brain damage (Neto et al., 2005). BCCAO and 2VAO can be combined in a model called four-vessel occlusion (4VO), which is a much more severe stroke (Akdemir et al., 2014), particularly in the CA1 area of the hippocampus, due to blockage of all major blood flow (Stetler et al., 2014). While 4VO is used to study chronic effects of ischemia, including cognitive impairment and behavior results (Neto et al., 2005; Valério Romanini et al., 2013), 4VO also causes changes in the expression of adenosine receptors in the hippocampus (Zhou et al., 2004). Adenosine release might change with severity of stroke, but there have been no studies of spontaneous adenosine release with the severe ischemia models of 2VAO and 4VO.

The goal of this study was to examine the changes in spontaneous, transient adenosine in the CA1 region of hippocampus in several ischemia models such as BCCAO, 2VAO, and 4VO. During BCCAO, the mildest stroke model, the frequency of the spontaneous, transient events increased with no change in the average event adenosine concentration. With 2VAO, a permanent and severe ischemia model, both the frequency and the average event adenosine concentration decreased. Similarly, 4VO also showed a decrease in the frequency of adenosine events and the average event adenosine concentration. Overall, this study shows that there is no enhanced release in the number of spontaneous, transient adenosine in the hippocampus as the intensity of the stroke increases. As a result, there is less transient adenosine available to inhibit neurotransmission or recruit blood flow, and thus spontaneous, transient adenosine changes do not play a protective role during intense ischemia in the CA1 area of hippocampus.

Materials & Methods

Materials

Sodium chloride (NaCl, 131.25 mM), sodium phosphate monohydrate (NaH2PO4, 10.0 mM, catalog number S9638) were purchased from Sigma Aldrich (St. Louis, MO, USA). Potassium chloride (KCl, 3.0 mM, catalog number P217), sodium sulfate (Na2SO4, anhydrous, 2.0 mM, catalog number S421), calcium chloride, (CaCl2, dihydrate, 1.2 mM, catalog number AC349610250), magnesium chloride (MgCl2, hexahydrate, 1.2 mM, catalog number BP214–500) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Phosphate buffered saline (PBS) at pH 7.4 was used for all in vitro measurements and was made using above mentioned chemicals. DMSO (dimethyl sulfoxide) was purchased from Amresco (Solon, OH, USA, catalog number 0231). Vascular occluders (catalog number VO1.5N) to induce the stroke were obtained from DOCXS biomedical (Ukiah, CA, USA). Bovie cauterizer kit was purchased from Fine scientific tools (Foster City, CA, USA, catalog number 18010–00).

Fast-scan Cyclic Voltammetry and Electrochemical Measurements

Carbon-fiber microelectrodes (CFMEs) were prepared as previously described (Huffman and Venton, 2008). Briefly, a carbon fiber (T-650, Cytec Engineering Materials, West Patterson, NJ, USA) of 7 μm in diameter was aspirated into a glass capillary (1.2 × 0.68 mm) and pulled using a vertical pipette puller (model PE-21; Narishige, Tokyo, Japan) into two electrodes. The exposed carbon fiber was cut to 125–150 μm with a scalpel. Electrical connection was made by backfilling the capillary with 1 M KCl. The silver-silver chloride reference electrodes were made in house by electrodepositing chloride onto a silver wire (Acros Organics, New Jersey, USA).

Fast-scan cyclic voltammetry (FSCV) was used to continuously monitor adenosine release on a sub-second time scale (Swamy and Venton, 2007). Adenosine was continuously monitored using FSCV and data were collected through computer controlled HDCV software (University of North Carolina, Chapel Hill, NC, USA) using a Dagan Chem Clamp potentiostat(Dagan Corporation, Minneapolis, MN, USA). The applied waveform was from –0.40 V to 1.45 V and back at 400 V/s vs Ag/AgCl reference, and was repeated for every 100 milliseconds. As this waveform produces a large background current, data were background subtracted (10 cyclic voltammograms averaged) to remove non-faradaic currents. Electrodes were post-calibrated with 1.0 μM adenosine in PBS solution, immediately following animal experiments and the average of triplicate current responses was used to estimate the transient adenosine concentrations in vivo.

Animal Experiments and Cerebral Ischemia and Reperfusion models

All animal experiments were in accordance with protocol number 3517 and approved by the Institutional Animal Care and Use Committee of the University of Virginia. Animal welfare was monitored daily by animal care staff. A graphical timeline is shown in Figure1 of the study design and typical experimental procedure in a flow chart. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA, RRID: RGD_734476) between 9–12 weeks/250–350 grams were housed in 12/12-hour light/dark cycles and fed ad libitum, and provided environmental enrichment. Animals were anesthetized with 1.5 g/kg of urethane intraperitoneally. Surgical areas were exposed by shaving around the surgical sites. This present study was not pre-registered. No randomization was performed to allocate subjects to specific stroke models in the study. Animals were assigned to different experimental groups by the experimenter without any blinding procedure. The experimenter decided the type of experiment on a given day in no specific order. At the conclusion of experiment, animals were injected with an excess dose of urethane and sacrificed through decapitation.

Figure 1. Graphical time-line of experimental procedure.

Figure 1.

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The experimenter decided the kind of stroke experiment or the control on a given day in no specific order. Rat was anesthetized using urethane anesthesia and the surgery was performed after the rat was fully anesthetized. Carbon fiber micro electrodes were implanted post-surgery and the spontaneous adenosine measurements were conducted as shown in the timeline above. For transmission electron microscope (TEM) imaging of the brains, rats were kept for 8 hours after the induction of the stroke. At the conclusion of the experiment, animal was killed by decapitation and a post-experiment calibration of the electrode was performed. The data were analyzed by automated software developed in our lab to find the spontaneous, adenosine events and then statistical analysis was performed.

Bilateral Common Carotid Artery Occlusion (BCCAO) model

To induce BCCAO, a midline incision was made under the neck to expose the tissue and the right common carotid artery was isolated and exposed from the vagus nerve and the cervical sympathetic chain. An occluder cuff was placed around the exposed artery and secured in place using a suture passed through the eyelet of the occluder. The same procedure was repeated on the other common carotid artery as well. Once both the occluders were secured in place, exposed neck area was sutured. The rat was then placed in a stereotaxic frame and 0.25 mL of bupivacaine (Sensorcaine, MPF, APP Pharmaceuticals, LLC; Schaumburg, IL, USA) was injected subcutaneously under the skin at the top of the skull prior to incision. Small burr holes were drilled in the skull for the placement of both working and reference electrodes (Paxinos and Watson, 2007). The carbon-fiber microelectrode was implanted in the hippocampus region (in mm from bregma): AP: −2.5, ML: +2.0, DV: –3.0. The Ag/AgCl reference electrode was placed on the contralateral side ~3 mm from midline. A temperature-controlled heating pad and thermistor probe regulated the rat’s body temperature (FHC; Bowdoin, ME, USA).

Two Vertebral Artery Occlusion (2VAO) model

Vertebral arteries were permanently occluded through electrocauterization (Neto et al., 2005; Jun Chen, Zao C. Xu, Xiao-Ming Xu, 2009). Prior to the electrocauterization, burr holes were drilled for the electrode placements as explained for the BCCAO model. The rat was placed in a stereotaxic frame and dorsal neck incision was made from the occipital bone to the second cervical vertebra (~2 cm in length). The surgical procedure was performed under a microscope and the paraspinal muscles were separated carefully to expose the alar foramen of the first cervical vertebrae. An electro cauterizer tip was pressed to each foramen to permanently cauterize the vertebral arteries on both sides.

Four Vessel Occlusion (4VO) model

The four vessel occlusion model is a combination of 2VAO and BCCAO (Neto et al., 2005; Chen et al., 2009; Wang et al., 2019). In a step wise process, first, occluders were placed on both sides of the common carotid arteries and burr holes were drilled for the electrode placements, as explained in BCCAO model. In the next step, vertebral arteries were permanently occluded as explained above in 2VAO model. Unlike many 4VO models in the literature,(Neto et al., 2005; Chen et al., 2009; Valério Romanini et al., 2013; Wang et al., 2019) we performed both surgeries at the same time, as our goal was to study early changes of spontaneous, transient adenosine immediately after the induction of ischemia.

Blood flow measurements

Cerebral blood flow was monitored by a laser Doppler flow meter with a 1.5 mm diameter probe (Model DRT4, Moor Instruments, Delaware, DE, USA). Anesthetized rat was placed in a stereotaxic frame, and burr holes were drilled in similar places as for the electrochemical experiments. The Doppler probe was placed over the hole in the skull in the cortex and blood flow recorded every 5 min under normoxia and I/R.

FSCV data collection and processing

Immediately after the conclusion of surgery, a carbon-fiber microelectrode was placed in the hippocampus region of the rat brain and equilibrated about 30 minutes until a steady background with optimal shape was observed. If spontaneous, transient adenosine release was not observed in the first 30 minutes of equilibration, a new electrode was inserted. Data were excluded if the frequency of transient adenosine release was less than ten events within 30 min. If robust spontaneous, transient adenosine release was not found, a new electrode was inserted, up to five new electrodes for each animal.

After optimization of electrode placement, data were collected for one to two hours of normoxia (pre-stroke). For BCCAO, ischemia was induced by inflating the occluders for 30 min and blood flow was completely blocked to both common carotid arteries. Then occluders were deflated, allowing the reperfusion and measurements were continued for 90 min. In control group of non-ischemia/sham experiments, occluders were placed but they were not inflated and 2 hours of data was collected. In 2VAO and 4VO models, electrocauterization of vertebral arteries must be performed before starting any electrochemical measurements (unlike BCCAO). In 2VAO model, adenosine was continuously monitored for 3 hours after the permanent occlusion (measurements typically started within one hour after occlusion). In 4VO, adenosine was measured for one hour after surgery (which is equivalent to 2VAO) and then BCCAO was induced (to make 4VO) for 30 min. Occluders were then released to allow reperfusion and measurements were continued for 90 min, but the vertebral arteries were still occluded.

Data analysis and Statistics

Spontaneous, transient adenosine occur randomly, lasting only 3 seconds on average. Spontaneous, transient adenosine events were identified and characterized using automated analysis programs, that identifies random adenosine events from FSCV data sets (Borman et al., 2017; Puthongkham et al., 2020). All statistics were performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). The data are presented as normalized mean ± SEM. A Kolmogorov-Smirnov (KS) test was used to determine underlying distributions between inter-event times (time between consecutive events). Averages were analyzed with one-way ANOVA, unpaired or paired t tests, depending on the number of variables in the data sets. p values <0.05 were considered significant. The study was exploratory and so predefined effects were not available, however sample sizes were chosen to be able to pick out 30% differences in means. For power analysis, the number of animals per group was calculated based on paired t-test compairson (using MedCalc). For cumulative concentration, the standard deviation of the difference between the normoxia and ischemia is 6 μM and the mean difference is 2.5. In order to see 20% difference with an α = 0.5 and power (1-β) of 0.8, n = 6 samples are needed. All data collected were used and no outliers were identified. All data were considered significant at the 95% confidence level. In this present study, 43 animals were included in the study, 42 were excluded based on the exclusion criteria and 16 animals were died during experiments, with a total of 101 animals were used.

RESULTS

Examination of blood flow and damage in different stroke models.

The goal of the study was to understand the temporal dynamics of spontaneous, transient adenosine changes under different stroke models in the CA1 region of hippocampus. The measurements were made in the CA1, since it is one of the most vulnerable brain regions to stroke (Marcoux et al., 1982). We compared three different ischemia models (1) Bilateral common carotid artery occlusion (BCCAO), (2) Two vertebral artery occlusion (2VAO), and (3) Four vessel occlusion (4VO) to understand changes in spontaneous, transient adenosine during varied stroke intensity. To confirm that different models could successfully induce stroke in the hippocampus, cerebral blood flow was measured using a laser Doppler probe during normoxia, ischemia, and reperfusion. While laser Doppler blood flow monitoring has been used during middle cerebral artery occlusion (MCAO) stroke models to monitor local blood flow and confirm a stroke (Cuccione et al., 2017; Ingberg et al., 2018), no one has compared the blood flow measurements for all three stroke models. Figure 2 shows the average blood flow in each stroke model. BCCAO results in an immediate 55% reduction of cerebral blood flow after the occlusion of carotid arteries. Releasing the occluders to allow reperfusion increased the blood flow to 83%, not quite back to baseline levels (Figure 2A). In the 2VAO model, blood flow dropped significantly to 68% of control after the permanent vertebral artery occlusion (Figure 2B). In the 4VO model, blood flow under normoxia was measured, the vertebral arteries occluded (reducing blood flow), and then common carotid arteries were occluded, which decreased blood flow to 53%. After releasing the occluders to allow the blood flow back through common carotid arteries, the blood flow increased near the levels of 2VAO to 64% (Figure 2C). Figure 2D shows the normalized blood flow measurements for all three stroke models. There was a significant overall effect on blood flow among the stroke models (One-way ANOVA, p<0.0001, n=3). Compared to normoxia, blood flow is significantly decreased during BCCAO ischemia and a further decreased during reperfusion (One-way ANOVA, p<0.0187, n=3). Normalized blood flow was also significantly different than normoxia for 2VAO, 4VO, and during the carotid arteries reperfusion for 4VO (One-way ANOVA, Dunnett’s test, p<0.0001, n=3).

Figure 2. Cerebral blood flow changes in rat brain.

Figure 2.

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Blood flow traces are individual replicates from separate animals. (A) Blood flow change during normoxia and I/R in BCCAO model. During normoxia, blood flow remained stable. Immediately after the induction of BCCAO, blood flow decreased (One-way ANOVA, p<0.0001, n=3) and during reperfusion, it increased but not back to baseline (One-way ANOVA, p<0.0187, n=3). (B) Blood flow in the 2VAO model. The initial 10 minutes of normoxia shows stable blood flow and immediately after the permanent electrocauterization of both vertebral arteries, blood flow dropped and stayed low (One-way ANOVA, p<0.0001, n=3). (C) Blood flow in 4VO model. After 10 minutes of normoxia, then 2VAO led to a sustained drop in the blood flow. After the induction of BCCAO, which is a 4VO stage, blood flow dropped further. When there was reperfusion of the carotid arteries, it returned almost to the levels of 2VAO (One-way ANOVA, p<0.0001, n=3). (D) Normalized blood flow in three stroke models. There was a significant main effect of group (One-way ANOVA, Dunnet’s test, p<0.0001, n=3). Statistical differences from normoxia (control) are marked on the graph. BCCAO-I is during ischemia, while when there is an R, it is during reperfusion (same for 4VO, where BCCAO reperfusion occurred during the time marked R).

To further investigate the pathological changes under these stroke models, we collected hippocampus brain sections and examined them using transmission electron microscopy (TEM). Brain samples were collected 8 hours after the induction of stroke, a sufficient time to see early damage. In a control sample, TEM data in Figure 3 showed no structural changes to cell nuclei (Figure 3A). Eight hours after 30 min of BCCAO, cells showed significantly smaller nuclei and exhibited peripheral condensation of chromatin (Figure 3B). The 2VAO sample, after 8 hours of permanent vertebral artery occlusion, showed higher condensation of chromatin, and greater swelling in the mitochondria with microbubbles formation (Figure 3C). The 4VO model, being the most severe of the three stroke models, caused more defined and larger swelling of mitochondria (Figure 3D).

Figure 3. TEM images of control, BCCAO, 2VAO, and 4VO model rat brain slices.

Figure 3.

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TEM images of the nucleus from rat brain hippocampus. Cerebral neurons in control rats showed normal cell nucleus and mitochondria (*). Neuronal mitochondria are classified as “not swollen” if the matrix (marked by *) is dark. Rats after 30 min of BCCAO and 8h of reperfusion led to substantial changes to organelle structure. After BCCAO, cell nucleus appears to have shrunken with microbubbles inside mitochondria. Rats after 8h of 2VAO appears to have condensed chromatin (arrow) and swollen mitochondria with more microbubbles (*). Rats after 1h of 2VAO and 30 min of BCCAO followed by 8h of reperfusion, showed extended swelling of mitochondria with much lighter (grey) matrix with microbubbles (*).

Control to ischemia models

As a control to all ischemia models, one group was given sham surgery, without cauterizing the vertebral arteries and not inflating the occluders around common carotid arteries. Adenosine was continuously monitored for 4 hours. The average number of events per hour were grouped into two bins that were each two hours and the number of events in1st 2 hours was significantly different compared to the 2nd 2 hours (paired t-test, p=0.0121, n=9, Figure 4A). There was a slight increase in the median inter-event time from 21 to 24 s between 1st 2 hours and 2nd 2 hours and a significant difference in the underlying distribution of transient adenosine frequency (KS test, p<0.0001, n=9, Figure 4B). There was no change in the average event adenosine concentration between the 1st and 2nd 2 hours (1st 2 hours: 0.12 ± 0.003 μM, n=1939 events, 2nd 2 hours: 0.11 ± 0.003 μM, n=1498 events, unpaired t-test, p=0.2119, Figure 4C). Also, there was no significant change in the cumulative adenosine concentration per hour from 1st 2 hours (12.8 ± 3.4 μM) to 2nd 2 hours (9.4 ± 3.2 μM) (paired t-test, p=0.133, n=9, Figure 4D). These control data are used as comparison data for the 2VAO and 4VO stroke models.

Figure 4. Control experiments with no ischemia.

Figure 4.

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(A) Number of spontaneous, transient adenosine events per hour changed between the 1st and 2nd 2 h periods (paired t-test, p = 0.0121). (B) Inter-event time of all adenosine events. There was significant difference between 1st (21s) and 2nd 2 hours (24s) (KS-test, p < 0.0001). (C) The average event adenosine concentration per transient. There was no significant change in the average event adenosine concentration per transient for 1st 2h and 2nd 2h (unpaired t-test, p = 0.2119, n= 1931, 1st 2h, n= 1490, 2nd 2h). (D) There was no significant change in the mean cumulative adenosine concentration between the 1st and 2nd 2 h periods (paired t-test, p = 0.133). All data and statistics are for n = 9 animals. Error bars are SEM.

Bilateral Common Carotid Artery Occlusion (BCCAO) model

For BCCAO, adenosine was measured during normoxia (2 hours) and ischemia/reperfusion (2 hours), so normoxia serves as within animal control, which allows paired statistics to be performed. There was no difference in the average number of spontaneous, transient adenosine changes per hour between normoxia and ischemia/reperfusion (paired t-test, p=0.4550, n=6) Figure 5A. However, the median inter-event time significantly increased from 14 s during normoxia to 28 s during ischemia and reperfusion (KS test, p<0.0001, n=6, Figure 5B). The average event adenosine concentration from all the animals did not change from normoxia to ischemia and reperfusion (normoxia: 0.13 ± 0.004 μM, n=971 events, I/R: 0.12 ± 0.003 μM, n=822 events, unpaired t-test, p=0.066 Figure 5C). The average cumulative adenosine concentration per hour also was not different between normoxia and ischemia/reperfusion (normoxia: 10.7 ± 5.5 μM, I/R: 8.2 ± 3.4 μM, paired t-test, p=0.3604, n=6 Figure 5D).

Figure 5. BCCAO model.

Figure 5.

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(A) Number of spontaneous, transient adenosine events per hour did not change between normoxia and I-R periods (paired t-test, p = 0.455). (B) Inter-event time. There was significant difference between normoxia (14s) and I-R periods (28s) (KS-test, p< 0.0001). (C) The average adenosine concentration per event. There was no significant change in the average event adenosine concentration for normoxia and I-R periods (unpaired t-test, p = 0.066, n= 971 events in normoxia, n= 822 events in I-R). (D) There was no significant change in the mean cumulative adenosine concentration between the normoxia and I-R periods (paired t-test, p = 0.3604). All data and statistics are for n = 6 animals. Error bars are SEM.

Two Vertebral Artery Occlusion (2VAO) model

In the 2VAO model, spontaneous, transient adenosine changes were continuously monitored after the permanent occlusion of both vertebral arteries for three hours. It is impossible to monitor adenosine prior to vertebral artery occlusion, since the entire surgery was performed under microscope. Due to the spatial limitations on the rat head, it is not possible to accommodate the electrode placement and microscope at the same time. Therefore, there is no within animal control for 2VAO model. The FSCV data collection started within one hour after 2VAO. Therefore, the data from the control group in Fig. 3 were used for comparison, and data are compared per hour, since the time course of experiments varied.

There was no significant difference in the average number of adenosine events per hour between control and 2VAO (unpaired t-test, p=0.3725, control, n=9, 2VAO, n=7) in Figure 6A. But the median inter-event time significantly increased from 22 s in controls to 30 s in 2VAO (KS test, p<0.0001, control, n=9, 2VAO, n=7, Figure 6B). The average event adenosine concentration from all animals also significantly decreased in 2VAO (control: 0.12 ± 0.002 μM, n=3421 events, 2VAO: 0.08 ± 0.002 μM, n=1567 events; unpaired t-test, p<0.0001 Figure 6C). The average cumulative adenosine concentration per hour did not significantly change between control and 2VAO (control: 11.1 ± 3.1 μM, n=9 to 2VAO: 6.5 ± 0.6 μM, n=7, unpaired t-test, p=0.2292, Figure 6D). Overall, the 2VAO model is a permanent occlusion of vertebral arteries and is more intense than the transient BCCAO. Even though there is large variance among animals, there is still a significant change in both the inter-event time and average event adenosine concentration, but not the number of events.

Figure 6. 2VAO model.

Figure 6.

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(A) Number of spontaneous, transient adenosine events per hour did not change between the normoxia and 2VAO periods (unpaired t-test, p = 0.3725, n=9 rats for control, n=7 rats for 2VAO). (B) Inter-event time. There was significant difference between normoxia (22s) and 2VAO (30s) periods (KS-test, p< 0.0001). (C) The average event adenosine concentration per transient. There was significant change in the average event adenosine concentration per transient for normoxia and 2VAO periods (unpaired t-test, p < 0.0001, n= 3421 events in control, n=1567 events in 2VAO). (D) There was no significant change in the mean cumulative adenosine concentration between the normoxia and 2VAO periods per hour (unpaired t-test, p = 0.2292, n=9 rats for control, n=7 rats for 2VAO). Error bars are SEM.

Four Vessel Occlusion (4VO) model

The 4VO model is the most severe stroke model among the three, since it is a combination of both 2VAO and BCCAO. In this model, vertebral arteries were first permanently occluded bilaterally (2VAO model) and spontaneous, transient adenosine changes were monitored for one hour, which is called the pre-occlusion period. In the second stage, the common carotid arteries were occluded bilaterally (BCCAO model) for 30 min, followed by 90 min of reperfusion by deflating the balloons. Similar to the 2VAO model, there is no within animal control.

The average number of events per hour was not significantly different between control and 4VO (unpaired t-test, p=0.0714, n=9, Figure 7A). However, the median inter-event time significantly increased from 22 s in control to 34 s in 4VO model (KS test, p<0.0001, n=9, Figure 7B). The average event adenosine concentration also significantly decreased from control to 4VO model (control: 0.12 ± 0.002 μM, n=3421 events, 4VO: 0.10 ± 0.003 μM, n=1096 events, unpaired t-test, p<0.0004, Figure 7C). However, the average cumulative adenosine concentration was not significantly different (control: 11.1 ± 3.1 μM to 4VO: 5.8 ± 1.7 μM, unpaired t-test, p=0.1560, n=9, Figure 7D). Overall 4VO model showed a significant change in both the inter-event time and average event adenosine concentration, but there is no enhanced release in the number of events.

Figure 7. 4VO model.

Figure 7.

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(A) Number of spontaneous, transient adenosine events per hour did not change between the normoxia and 4VO I-R periods (unpaired t-test, p = 0.0714). (B) Inter-event time. There was significant difference between normoxia (22s) and 4VO I-R periods (34s) per hour (KS-test, p< 0.0001). (C) The average event adenosine concentration per transient. There was significant change in the average event adenosine concentration per transient for normoxia and 4VO I-R periods (unpaired t-test, p < 0.004, n= 3421 events in control, n=1096 events in 4VO). (D) There was no significant change in the mean cumulative adenosine concentration between the normoxia and 4VO I-R periods per hour (unpaired t-test, p = 0.3348). All data and statistics are for n = 9 animals for control and 4VO. Error bars are SEM.

Comparison of stroke models

We compared all the ischemia models, since the overall goal of this study is to understand the time course changes of spontaneous, transient adenosine changes under different stroke models. There was no significant overall effect in the number of events per hour in all stroke models (One-way ANOVA, p=0.2105, Figure 8A). Figure 8B shows there is a significant main effect of stroke model on inter-event time (Kruskal-Wallis test, p<0.0001). All three stroke models showed overall significant difference in the inter-event time compared to control (One-way ANOVA, p<0.0001, Figure 8B). Thus, the frequency of spontaneous adenosine release is lower in the hippocampus during ischemia. All stroke models affect average event adenosine concentration and inter-event time, which uses data per transient for all the individual events and has more statistical power. Figure 8C compares average event adenosine concentration between the models and there is a significant main effect on concentration (One-way ANOVA, p<0.0001). BCCAO did not show any significant difference in the average event adenosine concentration compared to control (One-way ANOVA, Dunnett’s test p=0.0888) but both 2VAO and 4VO had significantly lower average event adenosine concentration than control (One-way ANOVA, Dunnett’s test, p<0.0001). But 4VO has significantly larger concentration than 2VAO (One-way ANOVA, Dunnett’s test, p=0.0124). Similar to the number of events, there was no significant change in the cumulative adenosine concentration per hour in all stroke models compared to control (One-way ANOVA, Dunnett’s test, p=0.3993, Figure 8D).

Figure 8. Comparison of ischemia models.

Figure 8.

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(A) Comparison of number of spontaneous, transient adenosine events in BCCAO I-R, 2VAO and 4VO I-R models to control. Number of spontaneous, transient adenosine events per hour did not change between control and three ischemia models (One-way ANOVA, Dunnet’s test). (B) Inter-event time, comparing BCCAO I-R, 2VAO and 4VO I-R models to control. All three models have a significant change in the inter-event time compared to control (One-way ANOVA, Dunnet’s test, p<0.0001). (C) Comparison of average event adenosine concentration per transient in BCCAO I-R, 2VAO and 4VO I-R models to control. BCCAO did not show significant difference (One-way ANOVA, Dunnet’s test, p=0.9921). Both 2VAO and 4VO I-R showed significant changes in the event adenosine concentration to control (One-way ANOVA, 2VAO, p<0.0001, 4VO, P=0.0024). 4VO I-R model showed significant change even when compared to 2VO model (One-way ANOVA, Dunnet’s test, 2VAO, p<0.0001, 4VO, P=0.0145). (D) There was no significant change in the mean cumulative adenosine concentration between control and all three stroke models (One-way ANOVA, Dunnet’s test).

DISCUSSION

In this study, we demonstrate decrease in spontaneous, transient adenosine frequency and concentration during cerebral ischemia in the hippocampus CA1. The main findings are that (1) there is an increase in inter-event time that correlates with increasing stroke and (2) the average event adenosine concentration decreased during 2VAO and 4VO, which are the more severe stroke models. During BCCAO, the mildest of the three models, transient adenosine event frequency decreased but the average event adenosine concentration did not change. However, both 2VAO and 4VO models showed a significant decrease in average frequency and concentration per event. Since there is no enhanced adenosine release, there could be a pool for spontaneous, transient adenosine that may be quickly depleted in the hippocampus during stroke, and thus there is less adenosine available for rapid neuroprotection. Future studies could examine the consequences of less spontaneous, transient adenosine signaling and whether it can be upregulated to provide more neuroprotection during ischemia in the hippocampus.

BCCAO: Changes in transient adenosine event frequency but not concentration

Global cerebral ischemia models, like BCCAO, are widely used to understand the changes in brain activity after ischemic insult (Dirnagl et al., 2009; Spray and Edvinsson, 2016). In the BCCAO model, ischemia and reperfusion were confirmed by blood flow measurements, as blood flow decreased by about 50% during ischemia and rose to about 80% of normal values after reperfusion. TEM data showed mild changes in brain pathology with a significantly smaller nuclei and peripheral condensation of chromatin, indicating BCCAO is a milder stroke model. Previous studies measuring every 20 min detected slow increases in adenosine during BCCAO, showing there could be a slow build up during acute BCCAO (Plaschke et al., 2001). Here, BCCAO caused a slower frequency of spontaneous, transient adenosine but no changes in the concentration of average event or the cumulative adenosine concentration. Thus, there is no increase in adenosine events signaling during BCCAO in the hippocampus.

The decrease in adenosine event frequency in the hippocampus during BCCAO contrasts with our previous study in the caudate, which found that the frequency of spontaneous, transient adenosine increased in the caudate (Ganesana and Venton, 2018). The baseline adenosine levels are comparable in these two brain regions (Pani et al., 2014) and average concentration of each transient event is similar, 138 nM in the hippocampus, and 140 nM in the caudate (Nguyen et al., 2014; Ganesana and Venton, 2018; Wang and Venton, 2019a). However, there are 25% more spontaneous adenosine events per hour in each animal in the hippocampus than in the caudate (number of events per hour, in caudate-putamen=77, n=7 rats; hippocampus=95, n=9 rats) (Ganesana and Venton, 2018). During BCCAO, the cumulative adenosine concentration increased by 53% in the caudate because the frequency of events increased. But in the hippocampus, the frequency decreased during BCCAO and the cumulative concentration trended down. The higher baseline frequency of spontaneous, transient adenosine signaling in hippocampus could mean that adenosine release is occurring at its maximum rate and the frequency cannot increase during ischemia. This study identifies important regional differences in spontaneous, transient adenosine signaling which may have profound effects for neuroprotection, as there is less adenosine available to provide transient neuroprotection in the hippocampus.

Two vertebral artery occlusion (2VAO) and 4VO show decreased frequency and concentration of spontaneous adenosine

In 2VAO, the vertebral arteries are permanently occluded, and blood flow dropped to 68% of control immediately and sustained at that level as reported previously (Wang et al., 2019). Previous histopathological studies showed that three days after the 2VAO, there were only a few surviving pyramidal neurons in the CA1 region of the hippocampus (Choi et al., 2007). Our TEM data 8 hours after 2VAO showed higher condensation of chromatin, and greater swelling in the mitochondria with the formation of microbubbles. 4VO caused blood flow to drop down to 50%, but after reperfusion of the carotid arteries, blood flow went back to 64%, which is close to the 2VAO level. Since 4VO is the most severe stroke model, TEM data showed extreme changes in mitochondrial swelling and more evident microbubble formation. A recent study of 4VO in a rat model showed that after 14 days, the nucleus structure of neurons in the CA1 area exhibited nuclear condensation, the cell arrangement was disorganized, and the number of normal neurons was small (Wang et al., 2019). While the previous literature reported on long-term effect of neuronal damage days after ischemia, here we show that early damage just 8 hours after ischemia also correlates with the severity of the stroke.

Due to the permanent nature of 2VAO, there was a large decrease in spontaneous, transient adenosine concentration and frequency compared to BCCAO. The median inter-event time was 30 s, which is 3 s more than the BCCAO model and 13 s more than the control. The average event adenosine concentration decreased from 117 nM in control to 87 nM in 2VAO, a 26% decrease. Therefore, 2VAO is a stronger stroke model than BCCAO and causes depletion of adenosine in the hippocampus.

Although, 4VO model is more severe than the 2VAO model, the results for adenosine events were very similar. The mean inter-event time increased to 34 s compared to 30 s for 2VAO. The average event adenosine concentration in 4VO model decreased significantly from control but was slightly higher at 100 nM than the 2VAO model of 87 nM. This could mainly be due to the transient ischemia and reperfusion phase from BCCAO, as blood flow was restored through the carotid arteries during the 90 min of reperfusion. The combination of BCCAO and 2VAO increased adenosine concentrations compared to 2VAO which may show that the more severe stroke does trigger some increases in adenosine compared to the milder stroke models alone. Overall, both 2VAO and 4VO are severe stroke models that result in both decreased frequency of adenosine events and decreased concentration of each adenosine event. But there is no enhanced release in the number of events. Thus, there is less spontaneous, transient adenosine available for neuromodulation in the hippocampus during early stages of these severe strokes.

Implications of lower adenosine availability during stroke in the hippocampus

Under normal conditions in the hippocampus, adenosine predominantly plays an inhibitory role due to high expression levels of the high-affinity A1R (Stockwell et al., 2017). A1 receptors can act as autoreceptors, regulating adenosine release (Cechova et al., 2010; Nguyen et al., 2014), and prolonged activation of adenosine A1 receptors in hippocampus causes endocytosis and subsequent synaptic depression (Chen et al., 2014). A1 receptors are upregulated in a 4VO model in the CA1 hippocampus after a short ischemic preconditioning (Zhou et al., 2004). Upregulation of A1 receptors would decrease the frequency of spontaneous adenosine release. Changes in expression of other receptors, such as NMDA, glutamate and GABA receptors also could change the frequency of spontaneous adenosine release (Nguyen et al., 2017). However, the time scale of this change in receptor levels is not known.

While many studies find adenosine slowly builds up during ischemia, (Plaschke et al., 2001) others have found decreases in adenosine and loss of neuroprotection after repeated exposures to hypoxia and MCAO (Cui et al., 2013). Using a combination of adenosine sensors and whole-cell recordings in the CA1 region, Pearson et al. found adenosine levels diminished during repeated exposure to hypoxia (Pearson et al., 2001). Thus, the releasable pool of adenosine decreased during stroke, which would reduce its ability to be neuroprotective. Our data for all ischemia models follows similar trends, showing that the releasable pool for spontaneous adenosine signaling might be going down since adenosine release is not enhanced in the hippocampus during severe ischemia. Adenosine is formed in the extracellular space primarily due to extracellular breakdown of ATP (Wang et al., 2020) and is likely released via exocytosis (Dale et al., 2000; Frenguelli et al., 2007; Corti et al., 2013; Lee and Venton, 2018). Thus, with severe ischemia in the hippocampus, there is likely less ATP being exocytotically released, perhaps due to its depletion under stress.

A recent study of transient BCCAO model (Wahul et al., 2018) found that hippocampus suffers a delayed and severe neuronal loss while cortex and striatum showed lesser, early losses. Specifically the CA1 region is highly vulnerable during global ischemia in animal models (Schmidt-Kastner and Freund, 1991; Bhuiyan et al., 2015) and humans (Bartsch et al., 2015), with neuronal death of CA1 neurons 48 to 72 hours after reperfusion. Thus, our findings of no enhanced release of adenosine could be a reason that hippocampus is susceptible to ischemic damage, as there would be less transient neuroprotection. Permanent ischemia models such as 2VAO and 4VO had higher depletion of adenosine as concentration of events decreased and also show more damage than transient BCCAO. More work is needed to examine the role of spontaneous adenosine in neuroprotection to understand if its depletion could be one of the main reasons for more damage.

The function of spontaneous, transient adenosine release has not been fully elucidated, but it is important in providing rapid neuromodulation. For example, spontaneous adenosine can transiently inhibit phasic dopamine neurotransmission, an effect that is reversible when adenosine is cleared (Ross and Venton, 2015). Spontaneous adenosine likely also transiently inhibits other neurotransmitters, such as glutamate, so reducing the amount of adenosine might lead to more neurotransmitter signaling and excitotoxicity. Adenosine also signals for immediate, transient increases in oxygen and blood flow, even during BCCAO (Wang and Venton, 2017, 2019b). Thus, the depletion of adenosine during ischemia in the hippocampus might cause less neuromodulation and treatments to upregulate adenosine or its receptors could be useful to retain neuronal function during ischemia. It is important to note that different pools of adenosine might exist in the cells and that extracellular adenosine might be released as adenosine or be formed from extracellular breakdown of ATP (Pearson et al., 2001). Our work shows that spontaneous adenosine signaling is not enhanced in the hippocampus during ischemia and this depletion might cause more neuronal damage compared to other areas, such as the caudate putamen, where depletion did not occur.

Conclusions

We found that spontaneous, transient adenosine event frequency did not increase in the CA1 region of hippocampus during severe global cerebral ischemia. We investigated three different ischemia models with varied severity and found that average event adenosine concentration goes down with severe and prolonged ischemic stroke. These findings lead to the existence of depletable adenosine pool and there is no enhanced release of spontaneous, transient adenosine in the hippocampus. Thus, we conclude that spontaneous, transient adenosine does not provide any rapid neuroprotective role during severe stroke models. Future studies could focus on identifying the role of adenosine pools to maintain spontaneous adenosine and provide a target for novel therapeutic approaches for neuroprotection during ischemia.

Acknowledgments and Conflict of Interest Disclosure

This research was supported by grants from NIH (R01NS076875 & R01EB026497) to BJV. The authors declare no competing financial interests.

All experiments were conducted in compliance with the ARRIVE guidelines.

Abbreviations:

FSCV

fast-scan cyclic voltammetry

BCCAO

bilateral common carotid artery

I-R

Cerebral ischemia and reperfusion

2VAO

Two vertebral artery occlusion

4VO

Four vessel occlusion

TEM

Transmission Electron Microscopy

ADO

Adenosine

RRID

Research Resource Identifiers

References:

  1. Akdemir G, Ratelade J, Asavapanumas N, Verkman AS (2014) Neuroprotective effect of aquaporin-4 deficiency in a mouse model of severe global cerebral ischemia produced by transient 4-vessel occlusion. Neurosci Lett 574:70–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartsch T, Döhring J, Reuter S, Finke C, Rohr A, Brauer H, Deuschl G, Jansen O (2015) Selective neuronal vulnerability of human hippocampal CA1 neurons: lesion evolution, temporal course, and pattern of hippocampal damage in diffusion-weighted MR imaging. J Cereb Blood Flow Metab 35:1836–1845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhattacharjee AK, White L, Chang L, Ma K, Harry GJ, Deutsch J, Rapoport SI (2012) Bilateral common carotid artery ligation transiently changes brain lipid metabolism in rats. Neurochem Res 37:1490–1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhuiyan MIH, Kim J-C, Hwang S-N, Lee M-Y, Kim SY (2015) Ischemic tolerance is associated with VEGF-C and VEGFR-3 signaling in the mouse hippocampus. Neuroscience 290:90–102. [DOI] [PubMed] [Google Scholar]
  5. Borman RP, Wang Y, Nguyen MD, Ganesana M, Lee ST, Venton BJ (2017) Automated Algorithm for Detection of Transient Adenosine Release. ACS Chem Neurosci 8:386–393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cechova S, Elsobky AM, Venton BJ (2010) A1 receptors self-regulate adenosine release in the striatum: Evidence of autoreceptor characteristics. Neuroscience 171:1006–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cechova S, Venton BJ (2008) Transient adenosine efflux in the rat caudate-putamen. J Neurochem 105:1253–1263. [DOI] [PubMed] [Google Scholar]
  8. Chen J, Xu XM, Xu ZC, Zhang JH (2009) Animal models of acute neurological injuries (Chen J, Xu ZC, Xu X-M, Zhang JH, eds). Totowa, NJ: Humana Press. [Google Scholar]
  9. Chen Z, Xiong C, Pancyr C, Stockwell J, Walz W, Cayabyab FS (2014) Prolonged Adenosine A1 Receptor Activation in Hypoxia and Pial Vessel Disruption Focal Cortical Ischemia Facilitates Clathrin-Mediated AMPA Receptor Endocytosis and Long-Lasting Synaptic Inhibition in Rat Hippocampal CA3-CA1 Synapses: Differential Regulat. J Neurosci 34:9621 LP–9643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choi Y-S, Cho K-O, Kim E-J, Sung K-W, Kim SY (2007) Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia. Exp Mol Med 39:556–563. [DOI] [PubMed] [Google Scholar]
  11. Corti F, Cellai L, Melani A, Donati C, Bruni P, Pedata F (2013) Adenosine is present in rat brain synaptic vesicles. Neuroreport 24:982–987. [DOI] [PubMed] [Google Scholar]
  12. Coyle P, Panzenbeck MJ (1990) Collateral development after carotid artery occlusion in Fischer 344 rats. Stroke 21 Available at: 10.1161/01.STR.21.2.316. [DOI] [PubMed] [Google Scholar]
  13. Cuccione E, Versace A, Cho T-H, Carone D, Berner L-P, Ong E, Rousseau D, Cai R, Monza L, Ferrarese C, Sganzerla EP, Berthezène Y, Nighoghossian N, Wiart M, Beretta S, Chauveau F (2017) Multi-site laser Doppler flowmetry for assessing collateral flow in experimental ischemic stroke: Validation of outcome prediction with acute MRI. J Cereb Blood Flow Metab 37:2159–2170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cui M, Bai X, Li T, Chen F, Dong Q, Zhao Y, Liu X (2013) Decreased extracellular adenosine levels lead to loss of hypoxia-induced neuroprotection after repeated episodes of exposure to hypoxia. PLoS One 8:e57065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dale N, Pearson T, Frenguelli BG (2000) Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice. J Physiol 526 Pt 1:143–155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dirnagl U, Becker K, Meisel A (2009) Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use. Lancet Neurol 8:398–412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frenguelli BG, Wigmore G, Llaudet E, Dale N (2007) Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus. J Neurochem 101:1400–1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ganesana M, Lee ST, Wang Y, Venton BJ (2017) Analytical Techniques in Neuroscience: Recent Advances in Imaging, Separation, and Electrochemical Methods. Anal Chem 89:314–341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ganesana M, Venton BJ (2018) Early changes in transient adenosine during cerebral ischemia and reperfusion injury. PLoS One 13:e0196932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gaur V, Aggarwal A, Kumar A (2009) Protective effect of naringin against ischemic reperfusion cerebral injury: Possible neurobehavioral, biochemical and cellular alterations in rat brain. Eur J Pharmacol 616:147–154. [DOI] [PubMed] [Google Scholar]
  21. Huffman ML, Venton BJ (2008) Electrochemical properties of different carbon-fiber microelectrodes using fast-scan cyclic voltammetry. Electroanalysis 20:2422–2428. [Google Scholar]
  22. Ingberg E, Dock H, Theodorsson E, Theodorsson A, Ström JO (2018) Effect of laser Doppler flowmetry and occlusion time on outcome variability and mortality in rat middle cerebral artery occlusion: inconclusive results. BMC Neurosci 19:24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Chen Jun, Xu Zao C., Xiao-Ming Xu JH (2009) Animal Models of Acute Neurological Injuries. In (Chen J, Xu ZC, Xu X-M, Zhang JH, eds), pp 1949–2456. Totowa, NJ: Humana Press. [Google Scholar]
  24. Lee ST, Venton BJ (2018) Regional Variations of Spontaneous, Transient Adenosine Release in Brain Slices. ACS Chem Neurosci 9:505–513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marcoux FW, Morawetz RB, Crowell RM, DeGirolami U, Halsey JHJ (1982) Differential regional vulnerability in transient focal cerebral ischemia. Stroke 13:339–346. [DOI] [PubMed] [Google Scholar]
  26. Melani A, Corti F, Stephan H, Müller CE, Donati C, Bruni P, Vannucchi MG, Pedata F (2012) Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum. Exp Neurol 233:193–204. [DOI] [PubMed] [Google Scholar]
  27. Melani A, Pugliese AM, Pedata F (2014) Chapter Thirteen - Adenosine Receptors in Cerebral Ischemia. In: Adenosine Receptors in Neurology and Psychiatry, pp 309–348. Academic Press. [DOI] [PubMed] [Google Scholar]
  28. Neto CJBF, Paganelli RA, Benetoli A, Lima KCM, Milani H(2005) Permanent, 3-stage, 4-vessel occlusion as a model of chronic and progressive brain hypoperfusion in rats: a neurohistological and behavioral analysis. Behav Brain Res 160:312–322. [DOI] [PubMed] [Google Scholar]
  29. Nguyen MD, Lee ST, Ross AE, Ryals M, Choudhry VI, Venton BJ (2014) Characterization of spontaneous, transient adenosine release in the caudate-putamen and prefrontal cortex. PLoS One 9:e87165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nguyen MD, Ross AE, Ryals M, Lee ST, Venton BJ (2015) Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism. Pharmacol Res Perspect 3:1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nguyen MD, Wang Y, Ganesana M, Venton BJ (2017) Transient Adenosine Release Is Modulated by NMDA and GABAB Receptors. ACS Chem Neurosci 8:376–385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pani AK, Jiao Y, Sample KJ, Smeyne RJ (2014) Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice Tansey MG, ed. PLoS One 9:e92422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Amsterdam-Boston: Elsevier Academic Press. [Google Scholar]
  34. Pearson T, Nuritova F, Caldwell D, Dale N, Frenguelli BG (2001) A depletable pool of adenosine in area CA1 of the rat hippocampus. JNeurosci 21:2298–2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pedata F, Gianfriddo M, Turchi D, Melani A (2005) The protective effect of adenosine A2A receptor antagonism in cerebral ischemia. Neurol Res 27:169–174. [DOI] [PubMed] [Google Scholar]
  36. Pedata F, Melani A, Pugliese AM, Coppi E, Cipriani S, Traini C (2007) The role of ATP and adenosine in the brain under normoxic and ischemic conditions. Purinergic Signal 3:299–310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Plaschke K, Grant M, Weigand MA, Züchner J, Martin E, Bardenheuer HJ (2001) Neuromodulatory effect of propentofylline on rat brain under acute and long-term hypoperfusion. Br J Pharmacol 133:107–116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Puthongkham P, Rocha J, Borgus JR, Ganesana M, Wang Y, Chang Y, Gahlmann A, Venton BJ (2020) Structural Similarity Image Analysis for Detection of Adenosine and Dopamine in Fast-Scan Cyclic Voltammetry Color Plots. Anal Chem 92:10485–10494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ross AE, Venton BJ (2015) Adenosine transiently modulates stimulated dopamine release in the caudate-putamen via A1 receptors. J Neurochem 132:51–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schmidt-Kastner R, Freund TF (1991) Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 40:599–636. [DOI] [PubMed] [Google Scholar]
  41. Serra PM, Boi M, Poddighe L, Melis T, Lai Y, Carta G, Quartu M (2019) Resveratrol Regulates BDNF, trkB, PSA-NCAM, and Arc Expression in the Rat Cerebral Cortex after Bilateral Common Carotid Artery Occlusion and Reperfusion. Nutr 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seydyousefi M, Moghanlou AE, Metz GAS, Gursoy R, Faghfoori MH, Mirghani SJ, Faghfoori Z (2019) Exogenous adenosine facilitates neuroprotection and functional recovery following cerebral ischemia in rats. Brain Res Bull 153:250–256. [DOI] [PubMed] [Google Scholar]
  43. Shin M, Wang Y, Borgus JR, Venton BJ (2019) Electrochemistry at the Synapse. Annu Rev Anal Chem 12:297–321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spray S, Edvinsson L (2016) Improved assessment of outcomes following transient global cerebral ischemia in mice. Exp Brain Res 234:1925–1934. [DOI] [PubMed] [Google Scholar]
  45. Stetler RA, Leak RK, Gan Y, Li P, Zhang F, Hu X, Jing Z, Chen J, Zigmond MJ, Gao Y (2014) Preconditioning provides neuroprotection in models of CNS disease: paradigms and clinical significance. Prog Neurobiol 114:58–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stockwell J, Jakova E, Cayabyab FS (2017) Adenosine A1 and A2A Receptors in the Brain: Current Research and Their Role in Neurodegeneration. Molecules 22:676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Swamy BEK, Venton BJ (2007) Subsecond detection of physiological adenosine concentrations using fast-scan cyclic voltammetry. Anal Chem 79:744–750. [DOI] [PubMed] [Google Scholar]
  48. Valério Romanini C, Dias Fiuza Ferreira E, Correia Bacarin C, Verussa MH, Weffort de Oliveira RM, Milani H (2013) Neurohistological and behavioral changes following the four-vessel occlusion/internal carotid artery model of chronic cerebral hypoperfusion: Comparison between normotensive and spontaneously hypertensive rats. Behav Brain Res 252:214–221. [DOI] [PubMed] [Google Scholar]
  49. Wahul AB, Joshi PC, Kumar A, Chakravarty S (2018) Transient global cerebral ischemia differentially affects cortex, striatum and hippocampus in Bilateral Common Carotid Arterial occlusion (BCCAo) mouse model. J Chem Neuroanat 92:1–15. [DOI] [PubMed] [Google Scholar]
  50. Wang W, Liu X, Lu H, Liu L, Wang Y, Yu Y, Zhang T (2019) A method for predicting the success of Pulsinell’s four-vessel occlusion rat model by LDF monitoring of cerebral blood flow decline. J Neurosci Methods 328:108439. [DOI] [PubMed] [Google Scholar]
  51. Wang Y, Copeland J, Shin M, Chang Y, Venton BJ (2020) CD73 or CD39 deletion reveals different mechanisms of formation for spontaneous and mechanically stimulated adenosine and sex specific compensations in ATP degradation. ACS Chem Neurosci 11:919–928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang Y, Venton BJ (2017) Correlation of transient adenosine release and oxygen changes in the caudate-putamen. J Neurochem 140:13–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang Y, Venton BJ (2019a) Comparison of spontaneous and mechanically-stimulated adenosine release in mice. Neurochem Int 124:46–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang Y, Venton BJ (2019b) Caffeine Modulates Spontaneous Adenosine and Oxygen Changes during Ischemia and Reperfusion. ACS Chem Neurosci 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xu C, Wu F, Yu P, Mao L (2019) In Vivo Electrochemical Sensors for Neurochemicals: Recent Update. ACS Sensors 4:3102–3118. [DOI] [PubMed] [Google Scholar]
  56. Zhou AM, Bin Li W, Li QJ, Liu HQ, Feng RF, Zhao HG (2004) A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats. Neurosci Res 48:397–404. [DOI] [PubMed] [Google Scholar]

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