Exposure to hypoxic-ischemic (HI) insults in newborns can predispose them to severe neurological sequela. The mechanisms underlying HI-related brain injury have not been completely elucidated. The neurovascular unit (NVU) is a composite of structures that protect the brain from the influx of detrimental molecules. Changes in the NVU after HI are important because they could reveal endogenous neuroprotective pathways in the cerebral microvasculature. Furthermore, the time course of changes in the NVU after exposure to HI in the newborn remains to be determined. In this study, we examined the effects of severe HI on the time course of changes in the NVU in neonatal rats. Brains were collected from rats exposed to right carotid artery ligation and 2 h of hypoxia on postnatal day 7 with recovery for 6 or 48 h after exposure to sham treatment (Sham) or HI. The right HI and left hypoxic alone sides of the brains were examined by quantitative immunohistochemistry for vascular density (laminin), pericyte vascular coverage (PDGFRβ), astrocyte vascular coverage (GFAP), and claudin-5 expression in the microvasculature of the cerebral cortex, white matter, and hippocampus. HI-related brain injury in neonatal rats was associated with increases in vascular density in the cortex and hippocampus 48 h after HI as well as neurovascular remodeling, including loss of pericyte coverage in the cortex and increases in claudin-5 in the hippocampus 6 h after HI. Astrocyte coverage was not affected by HI injury. The time course of the responses in the different components of the NVU varied after exposure to HI. There were also differential regional responses in the elements of the NVU in response to HI and hypoxia alone. Hypoxic-ischemic (HI) brain injury can cause significant damage resulting in long-term neurologic sequelae such as cerebral palsy, sensory and/or motor impairment, developmental delay, and cognitive abnormalities ,2. The pThe developing and adult brain are normally protected by a complex of barrier mechanism(s), which are termed the neurovascular unit (NVU) ,4. The NPrevious work has shown that ischemia in the fetus and HI in neonatal rodents can increase BBB permeability and disrupt the BBB in part by altering some components of the NVU ,6. StudiThe overall goal of the current study was to investigate the time course of changes in selected structural components of the NVU in neonatal rats after exposure to severe HI. We examined the density of blood vessels in the brain, pericyte and astrocyte vascular coverage of microvessels, and claudin-5 expression in microvessels after recovery for 6 and 48 h from HI induced by right carotid artery ligation and exposure to 8 percent oxygen for 2 h to understand the time course of changes in selected elements of the NVU after exposure to severe HI.p < 0.05) in microvascular density 48 h after HI compared with the Sham group. The laminin expression on the right HI side of the brain was also higher than on the left hypoxic side in the cerebral cortex 48 h after HI  immunostaining has been widely used as a molecular marker for pericytes in rodents . PDGFRβ GFAP  staining was used to visualize astrocytic vascular coverage in the cerebral cortex , white mClaudin-5 staining was used to visualize claudin-5 expression in the microvasculature as a representative of the tight junction proteins in the cerebral cortex , white mHI-related brain injury can result in severe morbidity and mortality in the newborn ,2,7,9. LThe major findings of the current study were as follows. First, microvessel vascular density was increased in the right HI cerebral cortex 48 h after exposure to HI compared with the Sham treated group and with the left cerebral cortex exposed to hypoxia alone. Second, the microvessel density also increased in the right hippocampus 48 h after exposure to HI compared with the left hippocampus exposed to hypoxia alone. Third, pericyte coverage was decreased in the right cerebral cortex 6 h after exposure to HI compared with the Sham treatment. However, there were no differences in pericyte coverage in the right cerebral cortex 48 h after HI compared with the Sham group or on the left side that was exposed to hypoxia alone. Fourth, there were no differences in pericyte coverage in white matter or hippocampus after HI or hypoxia alone. Fifth, there were no differences in astrocyte coverage in the cerebral cortex, white matter, or hippocampus after HI. Sixth, claudin-5 expression in the microvasculature increased in the left hypoxic and right HI hippocampus 6 h after the insults compared with the Sham group and also in the left hypoxic hippocampus 48 h after the insults. In addition, there were no differences in the vascular claudin-5 expression in the cerebral cortex or white matter after the insults.There is considerable evidence to suggest that pathophysiological events occurring during the perinatal period could alter the structure and function of the NVU . These pThe microvasculature quantification was determined with anti-Laminin antibodies. Laminin is an important molecular component of the vascular basal lamina, which has been extensively used as an important marker of the microvasculature in rodents ,11 and oIn the current study, microvessel density was increased 48 h after HI in the cerebral cortex and hippocampus. These findings are consistent with our previous work, in which we have demonstrated that the onset of neovascularization occurred between 48 and 72 h after brain ischemia in the cerebral cortex of the ovine fetus . NeurogePericytes are critical cellular components of the NVU that are central to the development and maintenance of the blood-brain barrier because of their contributions to the regulation of the endothelial cellular junctions ,26,27,28Astrocyte end-feet ensheathe the blood vessels in the brain and provide structural support to the cerebral vasculature . AstrocyMany published studies have previously used GFAP as a marker for astrocytes, including our own studies ,18,22,38In the current study, we showed that the astrocyte coverage was approximately 60–80% in the cerebral cortex, 70 to 80% in the white matter, and 70% to 90% in the hippocampus in the Sham control rats. There were no significant differences in the astrocyte coverage after exposure to HI or hypoxia or at the different time points in any of the brain regions after HI. Experimental studies regarding astrocytic responses to HIE or systemic LPS exposure in fetuses from various species suggested that astrocytes are relatively resistant to injury during the neonatal period and that the astrocytes adjacent to regions of necrosis are poised to proliferate . The curTight junctions (TJs) of the BBB are present between the endothelial cells of brain capillaries . There aRecently, there has been increasing evidence that alterations in the components of the NVU could be a reasonable source of biomarkers as indicators for brain injury after stroke in adult subjects . FurtherIn summary, the results of the current study appear to suggest that the NVU exhibits durability and regenerative capacity in response to HI injury in the neonate. We demonstrated that the pericytes might recover after HI injury and that astrocyte coverage was not significantly reduced after the injury, and claudin-5 expression was increased after the injury.There are several limitations to our study. Although we have identified the time course of changes in NVU components for the first time after HI injury in neonatal rats by immunohistochemical quantification, we were not able to complement these results by Western immunoblot analysis or enzyme-linked immunosorbent assay (ELIZA) on the brain samples from these studies because freshly frozen brain tissue was not available from the original studies. Moreover, we cannot comment upon sex-related differences due to the limited number of animals in the current study. In addition, we were not able to avoid the neuronal and nerve fiber staining, which could have been related to aldehyde fixative-induced auto-fluorescence because our samples required a long interval of fixation for optimal preservation after exposure to the 2 h of hypoxia-ischemia and the necessity to preserve the cellular and subcellular structures of the NVU. This could have affected the fluorescence quantitation.In conclusion, the current study revealed the time course of changes in NVU after HI-related brain injury in neonatal rat brains for the first time. HI injury was associated with increases in vascular density and neurovascular remodeling, including the loss of pericyte coverage in the cortex and increases in claudin-5 in the hippocampus, in neonatal rats. The time courses in the responses of the different components of the NVU varied after exposure to HI. There were also differential regional responses in the elements of the NVU in response to hypoxia and HI. We speculate that these alterations in the NVU could predispose to neuronal injury in the neonatal brain.The experimental procedures in this study were conducted after obtaining approval from the Institutional Animal Care and Use Committees of the Alpert Medical School of Brown University and Women and Infants Hospital of Rhode Island and in accordance with the National Institutes of Health Guidelines for the use of experimental animals. The brain tissue samples utilized in this study were residual samples obtained from neonatal rats enrolled in our previous report .The neonatal rats were born in time-mated dams  in the Animal Care Facility of Brown University. Pregnant Wistar dams were transported on embryonic day 15 (E15) or E16 and housed in a 12 h light/dark cycled facility with ad libitum access to food and water. The date upon which the rat pups were delivered was designated as postnatal day zero (P0). The pups from different litters born on the same day were culled and balanced so that each dam had no more than 10 pups. The pups were randomly assigned to the sham-operated control and HI-exposed groups on day 7 (P7). HI was induced by right carotid artery ligation along with exposure to 8% oxygen for 2 h according to the previously described methods ,51.n = 6 ), and in the group of neonatal rats at 6 h ) and 48 h ) after exposure to HI [Briefly, the pups were anesthetized with 3% to 4% isoflurane and maintained with 1% to 2% isoflurane during the procedure. The temperature of the pups was maintained at 36 °C during surgery with an isothermal heating pad. A midline skin incision was made on the neck overlying the trachea using sterile surgical scissors. The right common carotid artery (RCCA) was separated from the trachea and surrounding nerves and double ligated with 5-0 silk sutures. The incision was closed and sterilized with betadine and alcohol. Sham-treated subjects were exposed to the same procedure, except that the RCCA was not ligated. The pups were identified with a neonatal tattooing system . They were then returned to their dams for 1.5 to 3 h for feeding and recovery from the surgery and then were subsequently placed in a hypoxia chamber with 8% humidified oxygen and balanced nitrogen for 2 h with a constant temperature of 36 °C. The sham control-treated subjects were similarly exposed to room air for 2 h. The rats in each group were obtained from different litters to lower the potential for inter-litter variability. The animals in the Sham and HI groups were sacrificed at the same time points after exposure to sham HI or HI. The brains were obtained for the purpose of analysis from neonatal rats in the group of sham-operated control  or 48 h (HI-48 h: n = 5) or sham control treatment (Sham: n = 6). The pups were sedated with intraperitoneal injections containing ketamine (74 mg/kg) and xylazine (4 mg/kg). A hind leg pinch was performed after the injections to ensure that there was adequate sedation. The rat pups were perfused with cold saline and 4% paraformaldehyde (PFA) at a flow rate of 3 mL/min through the left ventricle as previously described [The brains were collected from rat pups after exposure to right carotid artery ligation and hypoxia (HI) with reoxygenation and recovery for 6 h  for antigen retrieval. Then, they were placed in a humidified chamber and blocked with Superblock T20 Blocking Buffer  for 2 h after three washes with PBS at 5 min intervals. The slides were then incubated overnight at 4 °C with appropriate primary antibodies. The primary antibodies were as follows: rabbit anti-laminin polyclonal antibody  applied at a dilution of 1:200, mouse anti-PDGFRβ monoclonal antibody  administered at a dilution of 1:10, chicken anti-GFAP monoclonal antibody  applied at a dilution of 1:500, and mouse anti-claudin-5 monoclonal antibody  applied at a dilution of 1:20. The next day, after three washes with PBS at 5 min intervals, the slides were incubated for 1 h with appropriate secondary antibodies. The secondary antibodies were as follows: biotinylated goat anti-rabbit antibody  applied at a dilution of 1:400, Alexa Fluor 594 goat anti-mouse antibody  applied at a dilution of 1:1000, and Alexa Fluor 594 goat anti-chicken antibody  applied at a dilution of 1:1000. Then, after three washes with PBS at 5 min intervals, the slides were incubated for 1 h with Streptavidin, Alexa Fluor 488 conjugate , which was applied at a dilution of 1:300. Finally, the immunoreactions were visualized with DAPI .An ORCA-Flash4.0 LT+ Digital CMOS camera  connected to the computer software Stereo Investigator 10.0  was used for image acquisition and subsequent quantification. Six fields from the cerebral cortex on each side of the brain were randomly selected and obtained at 10× magnification to determine the laminin density. Ten fields were collected at 40× magnification to examine vascular pericyte and astrocyte coverage and claudin-5 expression. In addition, two fields from the hippocampus from each side of the brain were randomly selected and obtained at 10× magnification to examine laminin density and seven at 40× magnification to examine vessel coverage and claudin-5 expression. Likewise, four fields from areas of white matter on each side of the brain were randomly selected and obtained at 10× magnification to determine the laminin density and five fields at 40× magnification were collected to examine vessel coverage and claudin-5 expression. The quantification of vessel density was performed according to the previously described methodology . BrieflyThe quantification of vascular coverage and claudin-5 expression was performed according to previously described methods . Brieflyp value of p < 0.05 was considered statistically significant.Values were presented as medians and scatter plots. Statistical analyses were performed using the Statistica  analysis program. The data were analyzed using a two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test when a significant difference was detected. A


To assess differences in examination scores among students with varying levels of testing anxiety, students were classified into low, moderate, or high testing anxiety groups based on their Cognitive Test Anxiety Scale–Second Edition (CTAS-2) score. Students were also stratified into two groups based on their cumulative grade point average (GPA). After transitioning to remote proctoring, examination scores significantly decreased for first-year (P1) students but significantly increased for second-year (P2) students. When stratified by CTAS-2 score, no significant difference in examination scores was found. When stratified by GPA, no significant difference in examination scores was found for P1 students, but a significant improvement was noted for P2 students with remote proctoring. The results of this study indicate that examination scores do not consistently improve or decline after introducing remote proctoring even when considering a student’s GPA and level of testing anxiety.Remote proctoring is often used to ensure testing integrity in a distance education environment but may impact academic performance. This quasi-experimental study aimed to evaluate changes in examination scores after transitioning to remote proctoring during the COVID-19 pandemic. Student pharmacists ( The COVID-19 pandemic has accelerated the pace of changes in education and learning and has forced educational institutions to rapidly transition out of the brick-and-mortar classroom and into a digital space ,3. WhileLive remote proctoring during remotely administered examinations has been proposed to be the most rigorous method to ensure academic integrity . As suchBy the end of the spring 2020 semester, students were reporting increased stress and anxiety levels. Given the previously published findings of increased stress and anxiety leading to worse academic performance in student pharmacists ,12 and mThis study aimed to evaluate the impact of a transition to remote proctoring on academic performance in first- (P1) and second-year (P2) Doctor of Pharmacy (PharmD) students. This study had two secondary aims: (1) to investigate whether academic performance after the transition to remote proctoring differed in cohorts of low, moderate, or high testing anxiety and (2) to examine whether effects on examination performance after transitioning to remote proctoring differed based upon the student’s overall academic performance, as indicated by cumulative grade point average (GPA).The remote proctoring process developed and implemented in this study only used internal resources and technology that were already available to the institution. Remote proctoring required student pharmacists to use two devices. On the first device, videoconferencing software  was used to monitor the student’s exam-taking space. On the second device, also known as the student’s testing device, the computer-based exam was administered via ExamSoft  in a secure manner. Secure exams in ExamSoft are set to restrict access to all other applications and disable all device functions during the examination.Proctors consisted of UTHSC College of Pharmacy faculty and staff who had completed a one-hour orientation and training session to ensure standardization of procedures amongst all proctors. This training session outlined proper procedures before, during, and after the examination along with a written guide and checklist for additional help. In addition, one-on-one sessions with IT staff were available to proctors if needed for adequate orientation to using the software and related technologies. Student pharmacists also attended a one-hour informational session to learn what to expect on examination day. Like proctors, student pharmacists were also able to consult with IT staff as needed before the first remotely administered and proctored examination.Prior to starting the examination, the proctor completed a check-in process with each student individually. This process included asking each student to show a 360° view of their workspace to ensure no unauthorized materials were present and an ID verification to ensure the correct identity of the exam taker. Throughout the session, students maintained audio and visual connections. Each proctor observed up to 24 students for the duration of the exam and noted potentially suspicious activities. Any suspicious activities were reported to the Office of Academic Affairs within the UTHSC College of Pharmacy. The UTHSC College of Pharmacy coordinator of testing was available to join the Zoom session and bring any student showing suspicious activities into a breakout room to actively intervene in real-time without interrupting other students. IT staff were available to help students and proctors with any technical difficulties during the examination.This quasi-experimental study evaluated composite examination scores before and after a transition to remote proctoring due to the COVID-19 pandemic. The study setting was a single 4-year, public PharmD program based in the Southeastern U.S. This study was reviewed by the Institutional Review Board and determined to be eligible for exempt review under 45 CFR 46.104(d)(1).The composite examinations are computer-based examinations that cover material from several different courses, occur at two-week intervals, and are administered in a three-hour timeframe throughout the five semesters of the didactic curriculum. The composite examination process has been previously described in greater detail . Table 1n = 192) and P2 (n = 195) students enrolled in spring 2020 at a single 4-year, public PharmD program were included and served as their own controls in the before-after analysis. Students were excluded from the analysis if they did not take any composite examinations with remote proctoring.All P1  scores that had been obtained previously were also obtained retrospectively.The CTAS-2 instrument includes 24 items to measure trait testing anxiety . CTAS-2 a priori level of significance of p < 0.05.All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 . Descriptive statistics were calculated for all variables. Shapiro–Wilk tests and scatterplots were examined to assess normality of the paired data in each cohort. Examination scores were all determined to be non-normally distributed for both the P1 and P2 cohorts, and thus, the appropriate non-parametric test was applied. Specifically, Wilcoxon signed rank tests compared median examination scores before and after the transition point. Kruskal-Wallis tests compared the change in composite examination scores amongst the CTAS-2 severity groups. Mann-Whitney tests compared the change in composite examination scores before and after the transition for the upper versus lower 50% of each cohort, as determined by cumulative GPA prior to the spring 2020 semester. All tests were two-tailed with an n = 2 in P1 cohort; n = 1 in P2 cohort) and thus had no participation in the remotely proctored examinations.A total of 190 P1 students and 194 P2 students were included in the analysis. Three students were excluded due to mid-semester attrition from the PharmD program (Demographic characteristics of participants are shown in p < 0.001). Conversely, composite examination scores significantly increased for P2 students after transitioning to remote proctoring (p < 0.001).p = 0.508). There were also no significant differences observed between the three CTAS-2 severity groups for P2 student pharmacists (p = 0.238).The change in examination performance after the transition to remote proctoring, with students stratified by CTAS-2 score, is depicted in p = 0.248). However, significant differences in examination scores after the transition to remote proctoring were observed when comparing the lower versus the upper 50th percentile GPA group for P2 students (p < 0.001). Specifically, the lower 50th percentile GPA group had a significant increase in composite examination score as compared to their upper 50th percentile GPA peers.Change in examination performance after the transition to remote proctoring, with student pharmacists stratified by GPA, is depicted in This study evaluated changes in academic performance among P1 and P2 students in the didactic PharmD curriculum at a single institution. Changes in academic performance were assessed by comparing examination performance prior to and after a transition to remote learning and proctoring due to the COVID-19 pandemic. The study results indicate that the impact of remote proctoring on examination scores may vary depending upon the student’s year within the curriculum as well as the student’s cumulative GPA. In this study, remote proctoring was not observed to significantly impact academic performance when comparing students with varying levels of testing anxiety.Our study contributes to the growing evidence regarding how remote proctoring during the COVID-19 pandemic may impact student academic performance. A study of undergraduate students enrolled in a remote psychopharmacology course during the pandemic in Brazil found that students with higher psychological distress scores were less likely to change from an incorrect to a correct answer . In our It has been previously reported that students with higher testing anxiety tend to perform worse on examinations that were proctored remotely as compared to those proctored and administered in person . In our When students were stratified by GPA in our study, no significant changes in academic performance after transitioning to remote proctoring were seen for P1 students; however, a significant improvement in academic performance was noted for P2 students with a GPA in the lower 50th percentile. Academic dishonesty is one possible explanation for the change in scores observed for P2 students. However, only a single instance of academic dishonesty was reported by a remote proctor during the remote proctoring period. A rigorous post-examination analysis was also conducted as part of the academic dishonesty detection process. This process included a hierarchical clustering analysis to detect similarity of student examination responses and an analysis of various data available from ExamSoft, including timestamped examination-taker activity and IP addresses for submissions and uploads .Our observations regarding impact on academic integrity are contrary to those of another study where ad hoc online testing implemented during the pandemic was found to have negative implications on academic integrity . A diffeAn alternative possible reason for the improvements in academic performance during the remote proctoring period that were observed for P2 students with a GPA in the lower 50th percentile is that P2 students may be more comfortable with the rigors of the program’s curriculum and the composite examination structure as compared to P1 students. Having more experience with these aspects of the PharmD program may have helped P2 students adapt with greater ease to the remote testing procedure and thereby perform better academically. In addition, P2 students may also be more accustomed to the technology used for distance education given their longer time of enrollment within the program. Our campus consists of three campuses split across two adjacent time zones, and remote learning technology, including Zoom, is used regularly to facilitate learning, even prior to the pandemic. Technology and a student’s familiarity with it has been reported to be a major aspect influencing student learning during the pandemic .Student preference for in-person versus remote proctoring is another factor to consider when interpreting our study results. A survey of students in medicine, dentistry, pharmacy, nursing, and applied medical sciences during the COVID-19 pandemic found that approximately one-third of students preferred remote examinations over in-person, and preference for remote examinations was significantly associated with GPA . Our stun = 2031) reported that less than half of participants were able to cope adequately with stress related to the COVID-19 pandemic [Evidence of stress and anxiety due to the pandemic itself has been previously demonstrated in both the general population and student pharmacists ,25,26 anpandemic . Howeverpandemic . In our There are limitations to this study to consider. One such limitation lies within the data analyzed for the P2 students. The spring semester for P2 students includes two modular courses that integrate pharmacotherapeutics, medicinal chemistry, and pharmacology: infectious disease (ID) and surgery, critical care, and transplant (SCCT). ID occurs in the first half of the semester and is assessed on examinations 1–5, while SCCT is in the latter half of the semester and assessed on examinations 6 through 8. Students may have found one modular course to be more challenging than the other, which may limit the ability to compare scores before and after the transition to remote proctoring around examination 5. However, the historical performance in these two courses has been comparable to performance included in this analysis. A second limitation is the study’s generalizability given that only a single PharmD program was included. The study sample mirrors the overall population of student pharmacists in the U.S., which is 63% female and 16.2% minority, supporting that this data is generalizable.Lastly, confounding factors may exist. One such factor to consider is the simultaneous change to remote instruction in spring 2020. However, course and examination content as well as examination construction remained the same with the transition to remote instruction. The COVID-19 pandemic has led to a heightened awareness of the “digital divide” in many areas, including education ,30,31. TAdditional studies should be conducted to further investigate the potential impacts of remote proctoring modalities and a student’s level of testing anxiety. Data from other PharmD programs are needed to evaluate remote proctoring effects on pharmacy student performance. Beyond the didactic curriculum, further research may consider examining experiential education performance. This may include exploring differences in learning and academic performance for student pharmacists who completed the didactic curriculum with assessments delivered in a remote manner as compared to those students who did not complete their didactic curriculum during the global pandemic.This study indicates that examination scores do not consistently improve or decline in student pharmacists after introducing remote proctoring. Furthermore, these findings suggest that remote proctoring procedures may impact students differently depending upon their year within the program. Remote proctoring was not observed to impact academic performance amongst students with varying levels of testing anxiety; however, a significant increase in score with remote proctoring was noted for P2 students within the lower 50th percentile GPA group, but no change was noted for P1s when stratified by GPA. Additional studies are needed to further characterize the impact of remote proctoring practices on academic performance in pharmacy education.