Post cardiac arrest physical exercise mitigates cell death in the septal and thalamic nuclei and ameliorates contextual fear conditioning deficits in rats

Abstract

A major concern for cardiac arrest (CA) survivors is the manifestation of long-term cognitive impairments. Physical exercise (PE) is a well-established approach to improve cognitive functions under certain pathological conditions. We previously showed that PE post-CA mitigates cognitive deficits, but the underlying mechanisms remain unknown. To define neuroprotective mechanisms, we analyzed whether PE post-CA protects neurons involved in memory. We first performed a contextual fear conditioning (CFC) test to confirm that PE post-CA preserves memory in rats. We then conducted a cell-count analysis and determined the number of live cells in the hippocampus, and septal and thalamic nuclei, all areas involved in cognitive functions. Lastly, we performed RNA-seq to determine PE post-CA effect on gene expression. Following CA, exercised rats had preserved CFC memory than sham PE animals. Despite this outcome, PE post-CA did not protect hippocampal cells from dying. However, PE ameliorated cell death in septal and thalamic nuclei compared to sham PE animals, suggesting that these nuclei are crucial in mitigating cognitive decline post-CA. Interestingly, PE affected regulation of genes related to neuroinflammation, plasticity, and cell death. These findings reveal potential mechanisms whereby PE post-CA preserves cognitive functions by protecting septal and thalamic cells via gene regulation.

Introduction

More than 350,000 out-of-hospital cardiac arrest (OHCA) and 200,000 in-hospital cardiac arrest (IHCA) cases occur annually in the United States and nearly 90% and 75%, respectively, are fatal among people of all ages. ¹ Critical in the recovery of OHCA surviving patients, is that they experience severe cognitive impairments manifested predominantly as memory loss. Therefore, it is important to develop early and effective rehabilitation therapies to mitigate the myriad of neurological deficits experienced by CA survivors. Our laboratory is focused in defining the mechanisms of cognitive deficits following CA and in finding potential therapies that may ameliorate these deficits.

Convergent evidence from human and animal studies has demonstrated the beneficial effects of physical exercise (PE) on cognitive functions, such as learning and memory, as a result of neuroplasticity, even under neurodegenerative conditions.^2,3 In addition, PE exerts neuroprotective effects in ischemic stroke by decreasing neuronal damage, alleviating astrocyte and extracellular matrix dysfunction, and strengthening cerebral blood vessels. ⁴ Moreover, we previously showed that a short bout of PE after asphyxial cardiac arrest (ACA) or middle cerebral artery occlusion improves cognitive outcomes in rats. ⁵

Many laboratories, including our own, have been defining potential mechanisms by which PE leads to the amelioration of deficits in cerebral ischemia models. Based on our previous study that PE improves cognitive deficits following ACA, the main goal of this study was to define whether PE after ACA protects against cell death of brain areas involved in the memory cognitive network. Thus, we first performed a contextual fear conditioning (CFC) test to confirm our earlier finding that PE after ACA preserves memory function, and then conducted an extensive histopathological analysis to test the hypothesis that this PE-induced cognitive protection is linked to the preservation of limbic structures involved in learning and memory. For this purpose, we examined the number of live cells (NLC) in the hippocampus, septal nuclei, and thalamic nuclei in post-ACA exercised rats. Lastly, we performed RNA-seq with the aim of assessing genome-wide changes in hippocampal gene expression induced by PE post-ACA to identify putative specific genes that play a role in improving cognitive outcomes after PE.

Methods

Animals

All animal procedures were approved by the Animal Care and Use Committee of the University of Miami and carried out as per the Guide and Use of Laboratory Animals published by the National Institutes of Health. A total of 43 animals were used to conduct all experiments and they were randomly assigned as follows: a batch of 16 animals for CFC, a batch of 17 animals for histopathology, and a batch of 10 animals for RNA-seq. All animals were 9- to 10-week-old male Sprague-Dawley rats with approximate weights of 300 g. The rats were paired in cages and habituated to the laboratory setting for at least 5 days prior to surgery. Experiments were performed following the ARRIVE guidelines.

Handling and training

All animals received 2 days of handling and training (H&T) prior to surgery (Figure 1) to become habituated to the environmental conditions of the upcoming PE sessions, ⁵ which consisted of a quiet room with ceiling lights off and a desk lamp illuminating the animals. During each H&T day, the rats were handled by an investigator using fabric gloves for 10 minutes and then walked on a treadmill (Columbus Instruments, Columbus, Ohio) at a speed of 5 m/min, where an electrical stimulation from the grid forced them to walk. Sham animals were placed on a stationary treadmill and were not forced to walk. Following the last session, the rats were placed in clean cages and fasted overnight for next morning surgery.

Figure 1.

Experimental timeline for all experiments. All animals received 2 days of H&T before undergoing surgery. Following 5 days of post-surgical recovery, the rats were randomly assigned to 5 consecutive days of either PE or sham PE. (a) After 10 days of post-exercise recovery, the rats were subjected to a 2-day CFC test to determine their cognitive functions and then euthanized. (b) After 3 days of post-exercise recovery, the rats were euthanized for histopathological analyses and (c) Following 30 min from the last PE session, the rats were euthanized for RNA-seq analysis.

Surgery

Cardiac arrest surgery was performed as previously described. ⁶ The rats were anesthetized with 4% isoflurane and a mixture of 30% oxygen and 70% nitrous oxide. Electrocardiographic leads were placed in the limbs to record EEG and the endotracheal was intubated. Endovascular access was accomplished by decreasing isoflurane to 1%. Once the animals were anesthetized, they were cannulated with a single lumen PE-50 catheter at the femoral artery to monitor blood pressure and gas while cannulation of the femoral vein was performed using a polyethylene catheter. Vecuronium (2 mg/kg) was administrated intravenously and isoflurane was then decreased to 0.5% along with mechanical ventilation (60 breaths/min). The ventilator from the endotracheal tube was then disconnected to cause asphyxial cardiac arrest (ACA) by apnea induction. Within a few minutes of asphyxia, the heart rate decreased, and the mean arterial blood pressure reached zero mmHg. At minute 8, minimal EEG activity was recorded, and resuscitation started by intravenous injection of epinephrine (5 µg/kg) and sodium bicarbonate (1 meq/kg). Immediately after, the animals received mechanical ventilation with 100% oxygen at a rate of 80 breaths/min and chest compressions until the heart restarted to pump. The ventilator was then lowered to 60 breaths/min and the oxygen dropped to 70%. After about 15 min of restored circulation, the catheters were taken out, and the endotracheal tube was removed followed by face-mask delivery of oxygen (1 liter/min). The wounds were sutured with 5-0 silk and the body temperature was kept at 37°C with heating lamps for 1 hour. Finally, the rats were accommodated in a humidified incubator for 24 hours at a temperature of 29°C, where they received 1.5 mL of normal saline an hour after the surgery. For the next two days, all rats were placed in individual cages and received buprenorphine twice daily (4 doses total) to control pain. The same surgical method, including isoflurane administration, was applied to Sham ACA animals with the exception of ACA induction.

Physical exercise

After 5 days of post-surgical recovery, all animals were placed back on the treadmill to conduct the PE sessions (Figure 1) with the same environmental conditions of the H&T. The treatments were performed between 6 p.m. and 7 p.m. every day for 5 consecutive days. Animals assigned for PE were forced to run by electrical stimulation at a speed of 15 m/min for 30 minutes. Sham animals were also placed on the treadmill (stationary) for 30 min, but without undergoing PE. Exercised animals were monitored for any physical impairment that would impede adequate running and unfit animals were excluded from the experiments. After their respective treatments, all rats were monitored for any injury and then placed individually in cages to allow recovery.

Contextual fear conditioning

Following 5 days of post-surgical recovery, the animals were divided into two groups of 8 rats, where one group received ACA and PE and the other group received ACA and Sham PE. 20 days after surgery, the rats were exposed to a 2-day CFC test (Figure 1(a)). The animals were placed in a conditioning apparatus (12″ W × 10″ D × 12″ H) inside a chamber (30″ W × 17.75″ D × 18.5″ H), which contained a stimulus light and 28 V exhaust fan (Coulbourn Instruments, Whitehall, PA). An electrical active gridded floor was connected to a precision animal shocker. The rats were first acclimatized in the fear conditioning room for 30 min and then placed inside the chamber. On the first day, at 340 seconds, the animals received a 2.0 mA shock for 2 seconds. The rats remained in the apparatus for 30 more seconds and then were transferred to their cages. On the second day, the rats were placed into the apparatus for 8 minutes, but no shock was delivered. A visual tracking software was used to analyze the freezing behavior as the percent of time spent frozen (FreezeFrame, Coulbourn Instruments). Index of fear memory was measured by subtracting freezing levels of day 1 from day 2 in each individual animal. Using our previous criteria, ⁵ animals presenting more than 50% freezing levels on the first day of the test were excluded from analysis (N = 1, PE group).

Histopathology

Following 5 days of post-surgical recovery, the animals were divided into three groups, the first group consisted of 5 animals that received ACA and PE, the second group consisted of 6 animals that received ACA and sham PE, and the third group consisted of 6 animals that experienced Sham ACA and Sham PE. 13 days after surgery, the animals were euthanized to conduct a histopathological cell-count analysis of the NLC (Figure 1(b)). The brain tissues were first sliced in blocks containing the nuclei of interest and then embedded in paraffin to be cut into 10 µm sections. The sections were stained with hematoxylin and eosin along with dehydration using increasing concentrations of alcohol. Once stained, the sections were mounted on a microscope and the images were scanned at 5× and analyzed at 40× magnifications using stereology with a MCID software (Imaging Research, Ontario, Canada). Cells that were not counted presented ischemic changes, including eosinophilic cytoplasm, dark-staining triangular shaped nuclei, and eosinophilic-staining nucleolus. As in previous studies, ⁷ the NLC in the CA1 hippocampal region were first counted in all animals that received ACA and compared to previous control animals. Following confirmation of ACA-induced cell death in CA1 region, the NLC were counted in multiple septal and thalamic nuclei.

Stereotaxic coordinates and nuclei distribution

The CA1 hippocampal region was located on Bregma section −3.80 mm. All septal nuclei were found on Bregma levels 0.2, 0.48, and 0.70 mm, while all the thalamic nuclei belonged to sections −1.60 and −1.40 mm from Bregma. The NLC were counted from sub-nuclei grouped as follows: Hippocampus: CA1 region; Lateral Septal nuclei (LSn): intermediate, ventral, and zona limitans parts; Medial Septal complex (MSc): medial septal, vertical diagonal band, and horizontal diagonal band; Anterior Thalamic nuclei (ATn): anteromedial, anteromedial ventral, anteroventral, anterodorsal, interanterimedial, interanteridorsal, and rhomboid nucleus areas; and Reticular Thalamic nucleus (RTn): reticular thalamus.

Stereological cell counting

The NLC in the hippocampus was counted in both sides of the entire CA1 region. For septal and thalamic histopathology, 7.5% of area sub fraction was counted and the total number of cells per section was calculated with this percentage. After taking the average number of cells of all sections and the average area outlined for cell count, the volumes for all sections were calculated. The septal nucleus involved 50 sections (∼500 mm³) while the thalamus areas comprised of 20 sections (∼200 mm³). The results were normalized as the number of cells in 1 mm³ and the averages for each, septal and thalamic regions, were taken to perform the statistical analysis.

RNA extraction

Following 5 days of post-surgical recovery, the animals were divided into two groups, where one group of 5 rats received ACA and PE and the other group of 5 rats received ACA and Sham PE. Every sample was run separately (not pooled). After 30 min from the last PE session, the rats were euthanized, and the whole hippocampus (CA1-3 and dentate gyrus) was harvested for RNA isolation (Figure 1(c)). The hippocampus was selected because it is among the most vulnerable brain regions to global cerebral ischemia-induced cell death. ⁸ Total RNA was extracted and purified using the Trizol/RNeasy hybrid method. Briefly, the starting material was homogenized using 1 mL Trizol reagent (Invitrogen), incubated at room temperature for 10 minutes, then chloroform was added (0.2 mL/mL Trizol used) and tubes were shaken vigorously for 20 seconds. After another 2-minute incubation at room temperature, samples were centrifuged for 20 minutes at 14,000 × rpm (4°C). The aqueous phase was then removed, placed into a new tube, combined with an equal volume of 70% EtOH and mixed gently by inversion, and loaded onto a RNeasy column. Subsequent purification of the RNA using the Qiagen RNeasy Mini Kit was done according to the manufacturer’s instructions (including DNase I treatment to remove remaining DNA). The concentration and integrity of isolated RNA were determined with Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and the Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA).

RNA-sequencing

RNA-seq was performed to assess genome-wide changes in hippocampal gene expression induced by PE post-ACA. Sequencing libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) KR1351 v2.17 (Roche, Indianapolis, IN) according to the manufacturer protocols. Paired-end sequencing was performed on the Illumina HiSeq3000 system (Illumina, San Diego, CA), yielding an average of 30 million reads/sample.

Raw FASTQ reads were processed by a bioinformatics pipeline including adapter trimming by TrimGalore (v0.4.2) (https://github.com/FelixKrueger/TrimGalore), alignment with the STAR aligner v2.5.0a to the rat reference genome (Rattus norvegicus, http://www.ensembl.org/index.html), and gene counts quantified using the GeneCounts module implemented in STAR. RNA-seq alignment quality control metrics were extracted with the CollectRnaSeqMetrics module. The gene counts for each sample were transformed and normalized using the variance-stabilizing transformation method implemented in the Bioconductor package DESeq2v2.2 in Rv3.4.3. Differential expression analysis was conducted using EdgeR, which models the over-dispersed Poisson count data using a negative binomial model and provides p-values from which estimates of false discovery rates (FDRs) are extracted using the Benjamini–Hochberg algorithm. ⁹ To select differentially expressed genes, a |log2FC| cutoff of >0.25 was used. Genevestigator (https://genevestigator.com/gv/index.jsp) and EnrichR (https://maayanlab.cloud/Enrichr/) software were used for categorization of genes altered by PE post-ACA.

DEG analysis between PE and resveratrol pre-conditioning

PE RNA-seq data set was compared to our previously published resveratrol pre-conditioning (RPC) RNA-seq data set ¹⁰ using Venny 2.1 from BioinfoGP and CNB-CSIC software. The complete list of the PE DEG was compared to our RPC DEG data set. The pathway analysis was examined using metascape [http://metascape.org] custom analysis ¹¹ using the following libraries from each of the categories: Functional Set: KEGG Functional Sets, GO Molecular; Functions Pathway: KEGG Pathway, GO Biological Processes, GeneGo Pathway, GeneGo Pathway Noodle, WikiPathways, PANTHER Pathway; Structural complex: KEGG Structural Complexes, GO cellular Components, Corum. The keyword “plasticity” was used for the membership analysis to determine whether the genes obtained from the two data sets are related to plasticity processes.

Real time qRT-PCR

Validation of the RNA-seq data was performed by real time qRT-PCR analysis (n = 5 per group) for 15 differentially regulated genes. Total RNA was extracted as described above and reverse transcribed into cDNA (1 μg input RNA) using the qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA). The cDNA was diluted five-fold prior to amplification. Quantitative real-time PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) was run on the QuantStudioTM 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA). All RT-qPCR reactions were set up in triplicate and β-actin was used as an endogenous housekeeping control gene. Primers were designed using Primer3 software (version 0.4.0).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 and RStudio software (R version 4.1.1) and personnel were blinded to experimental groups for all evaluations. CFC and histopathologic data obtained from these studies are presented as means with 95% confidence intervals (mean [95% CI]). Equality of variance and normality of residuals were tested using Levene's test and the Shapiro-Wilk test, respectively. All septal and thalamic histopathologic data were analyzed by one-way ANOVA followed by Dunnett's multiple-comparison test when appropriate and means represent NLC per mm³. Hippocampal data (non-normal) were analyzed using Kruskal-Wallis test and an unpaired Student’s t-test was used to evaluate CFC data. Statistical significance was defined as a p value <0.05.

Results

PE post-ACA preserves cognitive function 20 days after ischemic insult

As a first baseline study, we conducted a 2-day CFC test to examine the effect of PE post-ACA in cognitive functions. Specifically, the difference in freezing levels between the two days of the test were analyzed to assess fear memory. Confirming our previous study, we found a significant increase in the freezing behavior of rats that underwent PE after ACA compared to animals that were exposed to ACA but did not exercise (36.13 [12 to 60] and 8.52 [–0.15 to 17], respectively; N = 7–8; p < 0.05; see Figure 2).

Figure 2.

CFC performed 20 days following ACA. After 24 hours from the first session (shock delivery), the animals were placed back into the CFC apparatus for 480 seconds (no shock) to assess memory function. Bars show the difference in freezing levels as the percent of time spent frozen between day 1 and day 2 of animals in the ACA + PE group (N = 7) vs. animals in the ACA + Sham PE group (N = 8). Results showed a significant difference in freezing in the PE group compared to the sham PE group (p < 0.05). For statistical analysis, unpaired t test was used. *p < 0.05.

PE post-ACA does not protect CA1 hippocampal cells from dying

Based on these results, our next goal was to define if cognitive function preservation was linked to a histopathological cell-death reduction of the cognitive network involved in CFC. Since hippocampal CA1 cells are critically involved in the formation, consolidation, and retrieval of memories,^12,13 we conducted a histopathological cell-count analysis in the CA1 region to examine whether cells were protected by PE. We observed that PE post-ACA was unable to protect CA1 cells from dying 13 days following ischemia. Following ACA, a decrease in the number of live cells (NLC) was observed in exercised (368 [234 to 502]; N = 5) and non-exercised (345 [205 to 485]; N = 6) animals compared to Sham ACA animals (1,101 [1006 to 1196]; N = 6) (p < 0.05 and p < 0.01, respectively; see Figure 3). As a result, we reasoned that it is plausible that the improvement in memory function observed in the CFC test arises from protection in other limbic regions involved in memory. Therefore, we sought to analyze septal and thalamic nuclei, both areas known to have strong connections with the hippocampus.

Figure 3.

Cell survival in the CA1 hippocampal region after 13 days of ACA. Following ACA, the NLC in animals that did not exercise and animals that received PE were significantly lower compared to Sham ACA animals (p < 0.01 and p < 0.05, respectively). For statistical analysis, Kruskal-Wallis test was used. Horizontal scale bars represent 30 μm in length in the field of view of each representative image. *p < 0.05, **p < 0.01.

PE post-ACA mitigates cell death in the lateral septal nuclei and medial septal complex

The septal nuclei comprise lateral and medial groups that connect to other limbic areas, including the hippocampus and anterior thalamic nuclei, and are involved in memory.^14–18 The Lateral Septal nuclei (LSn) (Figure 4(a)) resulted in a statistically significant difference between groups as determined by one-way ANOVA (F(2,14) = 7.864, p < 0.01). Specifically, post Hoc comparison using Dunnett’s test indicated that the NLC was significantly lower in non-exercised post-ACA rats (18,100 [12,917 to 23,281]) compared to sham ACA animals (30,850 [24,101 to 36,602]) (N = 6 for both groups; p < 0.01); however, there was not a significant decrease in the NLC in exercised rats (25,680 [19,166 to 32,192]; N = 5) compared to sham ACA animals. Furthermore, one-way ANOVA (F(2,14) = 17.60, p < 0.001) also showed a statistically significant difference in the Medial Septal complex (MSc) between the groups ((Figure 4(b)), where Dunnett’s test for multiple comparison revealed a strong significant decrease in the NLC in non-exercised post-ACA rats (10,910 [9,699 to 12,112]) compared to sham ACA animals (20,275 [17,347 to 23,204]) (N = 6 for both groups; p < 0.0001). The post Hoc correction also revealed a significant decrease in the NLC in animals that received PE (15,460 [10,680 to 20,237]; N = 5) compared to sham ACA animals, but with a reduced statistical difference (p < 0.05).

Figure 4.

Cell survival in the lateral septal nuclei and medial septal complex after ACA. (a) LSn: animals that experienced ACA but not PE had a significantly lower NLC than Sham ACA animals (p < 0.01). The NLC was not significantly different between exercised rats and Sham ACA animals and (b) MSc: the NLC in animals that experienced ACA but not PE was lower than Sham ACA animals with a highly significant difference (p < 0.0001). Rats that received PE post-ACA also had a lower NLC than Sham ACA animals, but with a reduced significant difference (p < 0.05). Data are presented as means with 95% confidence intervals and statistical analyses were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Horizontal scale bars represent 30 μm in length in the field of view of each representative image. *p < 0.05, ****p < 0.0001.

PE post-ACA mitigates cell death in the anterior and reticular thalamic nuclei

The Anterior Thalamic nuclei (ATn) are involved in the hippocampal-diencephalic-cingulate circuits that support learning and memory ¹⁹ while the Reticular Thalamic nucleus (RTn) play important roles in modulating the flow of information of the other thalamic regions. ²⁰ The ATn histopathology (Figure 5(a)) showed no statistically significant differences between groups as determined by one-way ANOVA (F(2,14) = 3.340, p = 0.0652). The NLC was 35,012 [25,290 to 44,735] and 35,242 [30,956 to 39,527] for exercised (N = 5) and sham ACA animals (N = 6), respectively. Additionally, there was a strong trend of decreased NLC in non-exercised post-ACA rats (27,958 [23,479 to 32,437]; N = 6). Furthermore, one-way ANOVA revealed that there was a significant difference (F(2,14) = 3.815, p < 0.05) in the NLC between the groups in the RTn (Figure 5(b)). Dunnett’s test for multiple comparison found that the NLC was significantly lower in rats that experienced ACA but did not exercise (14,792 [6,754 to 22,831]) compared to sham ACA animals (23,143 [21,220 to 25,066]) (p < 0.05; N = 6 for both groups). However, there was not a significant difference in the NLC in exercised rats (21,891 [14,840 to 28,941]; N = 5) and sham ACA animals.

Figure 5.

Cell survival in the anterior and reticular thalamic nuclei after ACA. (a) ATn: the NLC was not significantly different between Sham ACA rats and exercised or non-exercised animals after ACA and (b) RTn: rats that did not exercise after ACA had a significantly lower NLC than Sham ACA animals (p < 0.05). There was not a significant difference in the NLC between exercised and Sham ACA animals. Data are presented as means with 95% confidence intervals and statistical analyses were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Horizontal scale bars represent 30 μm in length in the field of view of each representative image. *p < 0.05.

PE post-ACA initiates a signaling Cascade that promotes plasticity and transcriptional reprogramming

In view of our finding that PE significantly reduces cell death in limbic nuclei with reciprocal connectivity to the hippocampus, our next goal was to determine the effect of PE on gene expression following ACA in the hippocampus with the goal of defining putative molecular pathways associated with synaptic plasticity in the surviving hippocampal neurons. Following 30 min from the last treadmill session, rats were euthanized, and the hippocampus collected for RNA preparations. RNA-seq analysis (N = 5 for each group) resulted in 63 genes that were significantly upregulated and 32 genes that were significantly downregulated by PE post-ACA (p < 0.001; see Figure 6(a)). Validation of the RNA-seq data was performed by qRT-PCR analysis (N = 5) for 15 differentially regulated genes (Figure 6(b)).

Figure 6.

Gene regulation analysis following PE post-ACA. Animals were euthanized 10 days after ACA and hippocampal slices were obtained for RNA sequencing. (a) Exercised post-ACA animals showed a significant upregulation of 63 genes (p < 0.001) and significant downregulation of 32 genes (p < 0.001). (b) Validation of the RNA-seq data was performed by qRT-PCR analysis for 15 differentially regulated genes. (c) Approximately 40% of the genes differentially induced by PE post-ACA are linked to AD and 30% of the genes are also related to PE and (d) The z score was computed to assess deviations from the expected rank, derived from running the Fisher exact test for many random gene sets, and computing a mean rank and standard deviation from the expected rank for each term in the gene set library. The combined score was calculated by taking the log of the p-value from the Fisher exact test and multiplying that value by the z-score. The most significantly upregulated genes induced by PE post-ACA are related to neuroinflammation, plasticity (long-term memory, p = 0.00007), cell death and transcriptional regulation (regulation of transcription, p = 0.0004, and histone H3 deacetylation, p = 0.02).

PE induces upregulation of genes in the same direction as RPC model following ACA

Using the Venny comparison software, we found 12 DEG genes shared between the PE and RPC models: Arc​, Col1a1​, Dusp1,​ Fos,​ Junb,​ Kl,​ Nr4a1,​ Vwf,​ Kcne2,​ Aqp1,​ Zim1 and​ Npas4​. The pathway analysis gave a variety of possible pathways and several gene combinations. We restrict the searching parameters using the membership feature of metascape, using the keyword plasticity. Arc, cFos and Npas4 share this common pathway of regulation in the 12 DEG representing a 25% of the total list found between the PE and RPC RNA-seq data sets.

PE post-ACA affect expression of genes associated with alzheimer’s disease

To functionally categorize the genes altered by PE post-ACA, we analyzed our data with Genevestigator software. Unbiased analysis of the conditions that particularly affect the expression of the genes differentially induced by PE post-ACA identified Alzheimer’s disease (AD) as the most significant neurological disorder associated with our gene list. Approximately 40% of the genes differentially expressed by PE post-ACA have been linked to AD (Figure 6(c)) and 30% of the genes have also been shown to be related to PE (Figure 6(c)). There is a large overlap between the genes differentially expressed by PE post-ACA associated with AD and PE. To functionally categorize the differentially expressed genes, GO analysis was performed with the EnrichR software (Figure 6(d)). The most significantly enriched GO terms in the list of upregulated genes induced by PE post-ACA are related to neuroinflammation, plasticity (long-term memory, p < 0.001), cell death and transcriptional regulation (regulation of transcription, p < 0.001, and histone H3 deacetylation, p = 0.02).

Discussion

Our lab has previously demonstrated that short sessions of PE early post-ACA diminish cognitive deficits in rats; however, the mechanisms of this protection remained unknown. To delve into potential mechanisms by which PE may diminish cognitive deficits after ACA, we used the same experimental design from our previous study ⁵ to define whether PE post-ACA spares cell death in brain regions involved in modulation of cognitive functions and contextual memory.

Our CFC data confirmed our previous findings that 5 consecutive days of PE post-ACA improves cognitive outcomes 20 days following ischemia. Following ACA, exercised rats displayed higher freezing when transitioned from the first day to the second day of the behavioral test than non-exercised animals. This greater level of freezing suggests that rats that exercised after ACA were able to better remember and associate the environment with the aversive stimulus they received. ²¹

It is well established that the hippocampus is essential for the acquisition and expression of contextual conditioned fear memories.^12,13 Therefore, our hypothesis was that PE post-ACA would ameliorate hippocampal CA1 cell death. Nevertheless, our data showed that PE post-ACA was unable to rescue these cells from dying. We suggest that because the first PE session started 5 days after ACA, the effect of PE after this time interval was too late to rescue hippocampal cells due to their high vulnerability to ischemia.^22–24 In fact, our earlier study showed that CA1 pathology increases significantly between 3–7 days. ⁶ Nevertheless, it is important to note that we strictly measured CA1 cells in the dorsal hippocampal region because this is the most susceptible area following cardiac arrest. Therefore, there exists the possibility that PE has a different effect in other hippocampal regions.

Considering that PE post-ACA did not promote survival of CA1 hippocampal cells, we analyzed lateral and medial septal areas, which nuclei have strong connections with the hippocampus.^25,26 It is well established that the MSc are the nodal point for ascending afferent systems that result in the initiation of hippocampal theta oscillations.^27–29 Cholinergic and GABAergic cells involved in this mechanism contribute to spatial learning and exploratory behavior^30–32 and modulate the dynamics for encoding and retrieval of memories in the hippocampus. ³³ Moreover, the LSn play an important role in learning and memory ^17,34 and hippocampal inputs to these nuclei also involve a crucial gating mechanism for the expression of hippocampal theta oscillations. ³⁵

A key finding of our work was that ACA promotes cell death in the LSn and MSc. This was previously inferred in a study where numerous pyknotic cells were detected in the LSn after CA. ³⁶ We suggest that the loss of septal cells resulted in a decline of cognitive performance in our CFC test, as demonstrated in studies involving damaged septal areas.^37–39 The most striking finding in our study was that animals that underwent PE post-ACA showed a lower decrease in the NLC in the lateral septal nuclei and medial septal complex compared to animals that did not exercise after ACA. Considering the role of the LSn, we hypothesize that cells protected by PE in these nuclei are likely to maintain the mechanism that assists in normal contextual memory function. Moreover, it is possible that PE-induced MSc cell protection after ACA results in maintenance of synchronized theta rhythmicity in the hippocampus and thus preservation of contextual fear conditioning performance, even in a scenario of partial ACA induced-hippocampal cell death. Moreover, preservation of theta rhythmicity may also enhance plasticity in post-ACA surviving hippocampal cells and consequently improve memory during behavioral tasks.

Two other important brain regions involved in the memory network include the anterior and reticular thalamic nuclei. The ATn act as a hub between the hippocampus and prefrontal cortex to modulate theta rhythmicity within and between these two brain regions. ⁴⁰ It has been proposed that this connectivity play key roles in memory-guided attention ⁴¹ and the ATn-hippocampal circuit is involved in episodic memory, ⁴² including contextual memory. ⁴³ Furthermore, the RTn is the major inhibitory source between thalamic, comprising ATn, ⁴⁴ and cortical cells and thus regulate sensory processing, arousal, and cognition.^45–47 During quiescence, the RTn is also central generators of sleep spindles to promote memory consolidation.^45,48 Hence, sleep disruption, as seen in animals with damage in the RTn, ⁴⁹ is known to have drastic effects in memory function.^50,51

We found that ACA promotes pathology in the RTn, which is supported by a previous study that demonstrated that cell injury occurs in this nucleus following CA.⁵² Although not statistically significant, our data also showed that ACA results in a strong trend of decreased NLC in the ATn. Damage and atrophy of the ATn human brain has been reported following CA, causing memory impairment. ⁵³ Taking into account the large ATn volume we analyzed, composed of ventral, medial, intermedial and dorsal regions, ACA may have affected cell death only in some of these specific regions. Further studies would need to evaluate this by examining ATn areas individually. Furthermore, we also found PE-induced protection in both ATn and RTn areas. Considering that lesions in the ATn impair acquisition and retrieval of CFC due to loss of connection with the hippocampus,^43,54 it is reasonable to imply that loss of cells in some of these thalamic nuclei due to ACA results in poor CFC performance and the cell protection induced by PE attenuates this cognitive decline. Moreover, the ATn also innervate with other nuclei, such as the amygdala, cingulate cortex, mamillary body, and retrosplenial cortex, which associations are also critical for contextual fear memory.^42,55 Therefore, we suggest that activating these connections by PE may also counterbalance the loss of hippocampal-dependent function and result in enhanced cognitive performance when ATn cells are protected by PE following ACA. Furthermore, it is possible that damage to RTn after ACA would have resulted in disturbed consolidation of contextual fear memory during sleep transitioning from day 1 to day 2 in the CFC test, thus resulting in poor performance. Nonetheless, exercised rats may have properly consolidated shock-induced fear memory during sleep due to protected RTn cells, leading to enhanced cognitive functioning.

Interestingly, one of the main differences between the ATn and Rtn nuclei is the specific neuronal types that project to these areas. Although the RTn has in its majority cell bodies of GABAergic neurons, these cells receive inputs mainly from glutamatergic neurons. ⁵⁶ However, neuropils located at the ATn arise from a mixture of different neuronal types, including cholinergic neurons. ⁵⁷ Neuronal death caused by cerebral ischemia occurs for the most part because of excessive activation of glutamate receptors (excitotoxicity). Therefore, it is possible that the different results we observed between these two thalamic regions are due to the disparity of glutamatergic inputs.

Although PE was unable to ameliorate CA1 cell death post-ACA, it is plausible that surviving cells are more plastic after PE. Thus, to further elucidate the mechanisms by which PE protects and restores cognitive functions after ACA, we assessed genome-wide changes in hippocampal gene expression by RNA-seq. We observed that PE post-ACA significantly induced upregulation and downregulation of many genes, and we identified Alzheimer’s disease (AD) as the most significant neurological disorder associated with our gene list. AD shares several symptoms with ACA, such as deficits of cognitive abilities, and numerous studies have shown a significant decline of memory function, including contextual fear memory, in different models of AD.^58,59 Moreover, we found a large overlap between the differentially expressed genes (DEG) induced by PE post-ACA associated with AD and PE. Our functional categorization of the DEG showed that upregulated genes induced by PE post-ACA are related to neuroinflammation, plasticity (long-term memory), cell death and transcriptional regulation (regulation of transcription and histone H3 deacetylation). Examples of upregulated genes involved in neuroinflammation include Stat1 and Egr1, transcriptional activators of post-ischemic inflammation that are also known to be involved in AD neuropathology.^60,61 Upregulated plasticity genes, including Arc, are also suggested to participate in the pathogenesis of AD through amyloid-beta regulation of synaptic activity.^62,63 Arc is a master regulator for synaptic plasticity and stabilization of neuronal circuits; however, Arc expression is reduced in AD models affecting downstream genes involved in these functions. ⁶⁴ Thus, PE-induced upregulation of Arc supports our suggestion that post-ACA surviving hippocampal cells undergo plasticity, resulting in improved CFC performance. Furthermore, the most significant genes downregulated by PE post-ACA were involved in extracellular matrix organization. Taking everything into account, PE post-ACA is likely to result in preserved cognitive functions due to the initiation of various signaling cascades.

We carried out an in-depth pathway analysis by comparing our current results of post-ACA PE RNA-seq with other neuroprotection models. We conjectured that different models for neuroprotection share common pathways even if the neuroprotective strategy is different. Comparing DEG results found in the current PE model with the differential expression genes in our previous study using RPC, ¹⁰ there are at least 12 DEG in common and 3 of these genes participate in the regulation of synaptic plasticity. Despite the difference of the two models, fos, arc and npas4 are regulated in the same direction of expression in both models. These genes have been described to be associated with synaptic plasticity and neuroprotective effects in other models besides RPC.^65,66 Furthermore, enhancing synaptic plasticity is one of the mechanisms suggested to promote neuroprotection in pathologies, such as AD, where synaptic activity derangements are observed. ⁶⁷ Hence, it is possible that overexpression of these genes and the increased synaptic plasticity pathway induced by PE post-ACA led to a response with reduced cell death. Interestingly, in the gene list of genes differentially expressed in PE post-ACA there is a nominal enrichment (p = 0.052) of genes matching membership to the Kegg pathway: mmu04722: Neurotrophin signaling pathway, which was shown to be significantly induced by PE and enriched in the differentially expressed genes in the hippocampus of Fndc5 Knock-out mice. ⁶⁸ This suggest some convergence at the pathway level between the post-ACA PE induced transcriptome and the reported effects of PE that exceeds the genes induced by neuronal activity (such as Arc, Fos, Npas4, also shown to be differentially expressed in the hippocampus by PE ⁶⁹ )

In summary, our study confirms our previous finding that PE is a promising therapeutic approach for CA survivors. Importantly, we showed that following ACA, PE induces cell survival in septal and thalamic areas and promotes gene regulation and plasticity. These protective mechanisms plausibly result in the preservation of cognitive functions. A limitation to take into account is that only male rats were included in our analyses. Considering that sex dimorphism occurs in many aspects of the brain, including in experimental and clinical cases of ACA and stroke,^70–72 examining the effect of exercised post-ACA female rats would strengthen the current findings and these studies are underway in our laboratory. In addition, although we focused on memory as the main cognitive outcome of PE post-ACA, the multiple operations of the cells rescued by PE suggest that many other functions are probably preserved. Future studies will need to evaluate this by determining the specific cell types that are protected by PE post-ACA.