Athymic Nude Rats Research Articles

Hypertension and Cognitive Dysfunction in a Pregnant Rat Model of PE; a Role for CD4+ T Cells.

Preeclampsia (PE) is associated with hypertension (HTN) during pregnancy and activated CD4+ T cells, inflammatory cytokines, and autoantibodies to the angiotensin II type I receptor (AT1-AA). Having had COVID-19 (CV) during pregnancy is associated with an increased incidence of a PE-like phenotype. Both PE and CV have long-lasting neurological implications and studies show that nonpregnant COVID patients produce AT1-AA. We have shown that CD4+ T cells from PE women cause a PE phenotype in nude athymic rats. In this study, we sought to examine the role of CD4+ T cells from PE with a CV History (Hx) to contribute to a PE phenotype and to determine the importance of CD4+ T cells in cognitive dysfunction during pregnancy. At delivery, blood and placentas were collected, and one million placental CD4+ T cells from each PE and each normotensive patient, with (NT) or without (NP) a CV (Hx) during pregnancy, were isolated, purified, and injected i.p. into a gestational day (GD) 12 pregnant nude athymic rat (one patient/rat). At GD19, blood pressure (MAP) and circulating factors were assessed in recipient rats. Cognitive function and memory were assessed using Novel Object Recognition and Barnes Maze tests, respectively. Placental ACE-2 activity and AT1-AA were measured from COVID Hx patients. A one- or two-way ANOVA with Bonferroni's multiple comparisons test was used for statistical analysis. Blood pressure was increased in patients with PE, with or without COVID, compared to NT patients. There were no significant changes in placental ACE activity in patients with COVID Hx with or without PE. AT1-AA was elevated in PE patients and in both PE and NT COVID Hx compared to control NP. In pregnant recipient rats, MAP increased in CV Hx PE (113±2, n=8) compared to CV Hx NT (101±5, n=6). PE and PE CV Hx CD4+ T Cell recipient rats exhibited impaired memory and cognitive dysfunction (p < 0.05), compared to control groups. Recipient rats of PE CV Hx CD4+ T cells had elevated AT1-AA compared to NT CV Hx recipients. Both COVID Hx groups and recipients of PE CD4+ T cells had elevated TNF alpha compared to NP. Our findings indicate that pregnant patients with a Hx of COVID during pregnancy produce AT1-AA, with or without PE. Recipients of CD4+ T cells from PE with or without a CV Hx during pregnancy cause HTN and elevated AT1-AA. TNF-α is elevated in PE and in CV Hx NT and PE recipients. Interestingly, recipients of T cells from PE patients with or without a Hx of CV had worse cognitive function during pregnancy, compared to recipient rats of NP CD4+ T cells. These data demonstrate the importance of CD4+ T cells in HTN and impaired neurological function during PE in the presence or absence of a prior COVID-19 infection during pregnancy.

Abstract MP30: The Role of CD20+ B cells in Mediating Hypertension in Preeclampsia during Pregnancy

Pre-eclampsia (PE), new-onset hypertension after the 20 th week of pregnancy alongside other organ dysfunction, endothelial dysfunction, and immune activation. We have previously shown that CD19+ B cell adoptive transfer causes a PE phenotype in athymic nude rats. CD19 is present on all B cells including plasma cells. CD20 is absent from plasma cells but is present on mature B2 and memory B cells. The objective of this study was to determine if CD20+ B cells, excluding plasma cells, cause maternal hypertension, fetal growth restriction, and endothelial dysfunction. Three hundred thousand placental CD20+ B Cells from normal pregnant (NP) or PE patients were isolated by magnetic separation and injected i.p. into nude athymic rats on gestational day (GD) 12. On GD18, carotid catheters were inserted. On GD19, mean arterial pressure (MAP) was measured and blood and tissues were collected. Preproendothelin (PPET) expression was quantified in kidney and placental tissues using RT-PCR. A student’s t-test was used for statistical analysis. Participants whose placental B cells were used in this study had similar age, pre-pregnancy BMI, and diastolic BP on admission. PE participants had elevated systolic BP at admission (135±4 vs 120±4 mmHg) and delivery (139±6 vs 124±3 mmHg) as well as diastolic BP at delivery (79±4 vs 68±2 mmHg). PE participants also had trending decreases in fetal weight (2621±323 vs 3202±102 g) and gestational age (36±1 vs 39±0 weeks) at delivery. MAP increased from 107±4 mmHg (n=8) in the NP recipient rats to 123±2 mmHg (n=8, p&lt;0.01) in the PE recipient rats. Pup and placental weights were unchanged following adoptive transfer. Renal PPET expression increased by 2.595±0.61 fold (ΔΔct) in PE recipient rats (n=7, p&lt;0.05) compared to NP recipient rats (n=7). Placental PPET expression was unchanged following adoptive transfer. CD20+ B cells from women with PE contribute to hypertension and endothelial dysfunction during pregnancy. This data supports the important role of B cells in the development of PE and improves our understanding of the population of B cells responsible for contribution to the disease.

Abstract P193: Adoptive transfer of placental CD4+ T Cells from preeclamptic patients with a history of COVID-19 causes hypertension and cognitive dysfunction postpartum in a pregnant rat model of preeclampsia

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with activated CD4+ T cells, impaired cognitive function, and changes in cerebral blood flow (CBF) autoregulation. Previously, PE women have been shown to develop neurovascular and cardiovascular disorders earlier in life than those that had uneventful pregnancy. COVID-19 (CV) during pregnancy is associated with an increase incidence of the PE phenotype and is also associated with chronic neurovascular and cardiovascular consequences. Previously, we showed adoptive transfer of placental CD4+ T cells from PE women into athymic nude pregnant rats causes a PE phenotype, however we don’t know the role of CD4+ T cells to cause long-term neurovascular or cardiovascular consequences in PE in the presence or absence of a history (Hx) of COVID-19 during pregnancy. Therefore, we hypothesize that CD4+ T cells from PE patients with and without a Hx of CV causes hypertension and neurovascular dysfunction compared to CD4+ T cells from normotensive patients. One million placental CD4+ T cells from Normotensive (NT), CV Hx NT, and PE patients with or without a CV Hx were isolated and injected interperitoneally (i.p.) into pregnant nude athymic rats on gestational day (GD) 12. Dams were allowed to deliver and aged 12-weeks after delivery. At 12-weeks postpartum, blood pressure (MAP) was measured; CBF autoregulation was measured by laser Doppler flowmetry. The Barnes maze and the novel object recognition behavioral assays were used to assess cognitive function 12-weeks postpartum. A two-way ANOVA was used for statistical analysis. At 12-weeks postpartum, MAP increased in CV Hx PE (153±6, n=7, p&lt;0.05) and PE (150±5 mmHg, n=5, p&lt;0.05) compared to CV Hx NT (121±6, n=6) and NT (116±6 mmHg, n=6), respectively. Behavioral analysis showed CV Hx PE and PE exhibited impaired memory and learning (P&lt;0.05) compared to either NT group. Our findings indicate that rat recipients of CD4+ T cells from PE patients in the presence or absence of CV Hx have HTN and cognitive dysfunction in the postpartum period compared to recipients of control CD4 + T cells. These data indicate the importance of CD4+ T cells in long-term consequences of PE and COVID infection during pregnancy.

Extracellular vesicles from dental pulp mesenchymal stem cells modulate macrophage phenotype during acute and chronic cardiac inflammation in athymic nude rats with myocardial infarction

Background/aimsExtracellular vesicles (EVs) derived from dental pulp mesenchymal stem cells (DP-MSCs) are a promising therapeutic option for the treatment of myocardial ischemia. The aim of this study is to determine whether MSC-EVs could promote a pro-resolving environment in the heart by modulating macrophage populations.MethodsEVs derived from three independent biopsies of DP-MSCs (MSC-EVs) were isolated by tangential flow-filtration and size exclusion chromatography and were characterized by omics analyses. Biological processes associated with these molecules were analyzed using String and GeneCodis platforms. The immunomodulatory capacity of MSC-EVs to polarize macrophages towards a pro-resolving or M2-like phenotype was assessed by evaluating surface markers, cytokine production, and efferocytosis. The therapeutic potential of MSC-EVs was evaluated in an acute myocardial infarction (AMI) model in nude rats. Infarct size and the distribution of macrophage populations in the infarct area were evaluated 7 and 21 days after intramyocardial injection of MSC-EVs.ResultsLipidomic, proteomic, and miRNA-seq analysis of MSC-EVs revealed their association with biological processes involved in tissue regeneration and regulation of the immune system, among others. MSC-EVs promoted the differentiation of pro-inflammatory macrophages towards a pro-resolving phenotype, as evidenced by increased expression of M2 markers and decreased secretion of pro-inflammatory cytokines. Administration of MSC-EVs in rats with AMI limited the extent of the infarcted area at 7 and 21 days post-infarction. MSC-EV treatment also reduced the number of pro-inflammatory macrophages within the infarct area, promoting the resolution of inflammation.ConclusionEVs derived from DP-MSCs exhibited similar characteristics at the omics level irrespective of the biopsy from which they were derived. All MSC-EVs exerted effective pro-resolving responses in a rat model of AMI, indicating their potential as therapeutic agents for the treatment of inflammation associated with AMI.

Three-Dimensional Bio-Printed Tubular Tissue Using Dermal Fibroblast Cells as a New Tissue-Engineered Vascular Graft for Venous Replacement.

The current study was a preliminary evaluation of the feasibility and biologic features of three-dimensionally bio-printed tissue-engineered (3D bio-printed) vascular grafts comprising dermal fibroblast spheroids for venous replacement in rats and swine. The scaffold-free tubular tissue was made by the 3D bio-printer with normal human dermal fibroblasts. The tubular tissues were implanted into the infrarenal inferior vena cava of 4 male F344-rnu/rnu athymic nude rats and the short-term patency and histologic features were analyzed. A larger 3D bio-printed swine dermal fibroblast-derived prototype of tubular tissue was implanted into the right jugular vein of a swine and patency was evaluated at 4 weeks. The short-term patency rate was 100%. Immunohistochemistry analysis showed von Willebrand factor positivity on day 2, with more limited positivity observed on the luminal surface on day 5. Although the cross-sectional area of the wall differed significantly between preimplantation and days 2 and 5, suggesting swelling of the tubular tissue wall (both p < 0.01), the luminal diameter of the tubular tissues was not significantly altered during this period. The 3D bio-printed scaffold-free tubular tissues using human dermal or swine fibroblast spheroids may produce better tissue-engineered vascular grafts for venous replacement in rats or swine.

CD4+ T Cells from Women with Preeclampsia During Pregnancy Contribute to Hypertension, AT1-AA, and Neurovascular Dysfunction in a Pregnant Rat Model of PE

Objective: Preeclampsia (PE) is characterized by new onset hypertension (HTN) during pregnancy and is associated with activated CD4+ T cells and agonistic antibodies against the angiotensin II type 1 receptor (AT1-AA). Interestingly, having had COVID-19 during pregnancy is associated with increased incidence of a PE-like phenotype. Both PE and CV have long lasting neurological implications. We sought examine a role for CD4+ T cells from PE with or without a CV Hx to contribute to a PE phenotype and neurovascular dysfunction postpartum (PP). Methods: We utilized adoptive transfer of one million placental CD4+ T cells from PE and normotensive (NT) patients with or without a CV (Hx) during pregnancy injected i.p. into gestational day (GD) 12 nude athymic rats athymic and determined neurological consequences at 12 weeks PP. At GD19, blood pressure (MAP) and AT1-AA were measured. At 3 months PP, memory and error in navigation were assessed by Barnes maze consecutively for 5 days. CBF was measured by laser Doppler flowmetry. A two-way ANOVA was used for statistical analysis. Results: In pregnant recipient rats, MAP increased in CV Hx PE (113±2, n=8) and PE (115±2 mmHg, n=10) compared to CV Hx NT (104±4, n=7) and NT (98±2 mmHg, n=7 p&lt;0.05). AT1-AA increased in PE (8±2 ΔBPM, n=4) and CV Hx PE (4±2 ΔBPM, n=4) compared to NT (-3±2 ΔBPM, n=4, p&lt;0.05) and CV Hx NT (2±2 ΔBPM, n=2) respectively. At 3 months PP, PE and CV Hx PE had impaired CBF autoregulation compared to CV Hx NT and NT rats, and both PE groups exhibited greater mistakes and latency in maze navigation, however, the scores were worse in those receiving T cells from PE patients without a Hx of CV compared to all other groups. Conclusion: Our findings indicate that pregnant recipients of CD4+ T cells from PE with or without a CV Hx during pregnancy cause HTN and increased AT1-AA compared to recipients of NT or NT Hx CV-19 CD4 + T cells. Interestingly, recipients of T cells from PE patients without a Hx of CV had worse CBF and cognitive function at 3 months PP, demonstrating the importance of CD4+ T cells in HTN and impaired neurological function during pregnancy PP in patients with previous PE. This study was supported by NIH grants RO1HD067541 (BL), F31HD110230 (NC), P20GM121334 (BL, LMA), and American heart association (AHA) early career award 19CDA34670055 (LMA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of T cell stimulated agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) in mediating multiorgan dysfunction in IL-17 induced hypertension during pregnancy.

Preeclampsia (PE), new-onset hypertension during pregnancy accompanied by organ dysfunction, is associated with chronic inflammation including elevated IL-17, CD4+ T cells, B cells and natural killer (NK) cells. IL-17 can serve as a signal for either the adaptive or innate immune activation. We have previously shown that IL-17 contributes to increased blood pressure in association with elevated TH17 cells, NK cells and B cells secreting angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA) during pregnancy. Moreover, we have shown an important role for CD4+T cells and AT1-AA in multiorgan dysfunction as measured by mitochondrial oxidative stress (mt ROS). However, we do not know the role of adaptive immune cells such as T cells or B cells secreting AT1-AA in mediating the PE phenotype in response to elevated IL-17. In order to answer this question, we infused IL-17 (150 pg/day i.p.) into either Sprague Dawley (SD) or athymic nude rats via mini-osmotic pump from gestational day (GD) 14-19 of pregnancy. On GD 19, blood pressure was determined and NK cells, mtROS and respiration and AT1-AA production from B cells were measured. Infusion of IL-17 increased blood pressure in the presence or absence of T cells. Mean arterial pressure (MAP) increased with IL-17 from 98±2mm Hg (n=12) to 114±2 (n=12) in SD rats and from 99±4mm Hg (n=7) versus 115±2mm Hg (n=7) in athymic nude rats. Similar trends were seen in NK cells and placental mt ROS. Knowing that IL-17 stimulates AT1-AA in SD pregnant rats, we included a group of SD and athymic nude pregnant rats infused with IL-17 and the AT1-AA inhibitor peptide ('n7AAc'). The inhibitor attenuated blood pressure (104.9±3.2, p=.0001) and normalized NK cells and mt function in SD pregnant rats. Importantly, the AT1-AA was not produced in pregnant nude IL-17 treated rats, nor did 'n7AAc' effect MAP, in nude athymic rats. These findings suggest two conclusions; one is that IL-17 causes hypertension and multiorgan dysfunction in the absence of T cells and AT1-AA, possibly through its activation of innate cells and secondly, in the presence of T cells, blockade of the AT1-AA attenuates the effect of IL-17. This study indicates the critical effects of elevated IL-17 during pregnancy and suggest treatment modalities to consider for PE women.

205 Microglia-Mediated Survival Outcomes in Preoperative Radiation for Glioblastoma: Insights From a Preclinical Model

INTRODUCTION: Glioblastoma (GBM) is the most common and devastating primary brain tumor in adults. The current standard of care includes maximal safe resection followed by concurrent radiation and temozolomide-based chemotherapy. However, preoperative radiotherapy is used in different cancer types and commonly used for brain metastasis to shrink tumors, but it has never been used in for primary GBM. METHODS: Thirty-nine immunocompromised adult athymic nude female rats were orthotopically engrafted with a GBM1A patient-derived glioma cell line and randomized to either receive radiotherapy prior to tumor resection (RTRE) or postoperative radiotherapy (RERT). The rats received 30 Gy hypofractionated stereotactic radiation in 5 fractions of 6 Gy. Kaplan-Meier curve was used to plot the survival. Hematoxylin-eosin staining, microglia/macrophages using IBA-1 and TMEM119 markers were used in resected and recurrent tumor tissues. Moreover, nuclei area was measured on all tumor samples. Student’s T-test and analysis of variance (ANOVA) were used for statistical analysis. RESULTS: Kaplan-Meier analysis showed a significant survival benefit in the RTRE group, with median survival of 18.28 weeks compared to 14.85 weeks for RERT (p &lt; 0.001), which correlated with the time of recurrence. Simultaneously, an increase in the nuclei area was found in the resected tumor tissues from the RTRE group (p &lt; 0.001) and RERT recurrent tumor tissues (p &lt; 0.001). Interestingly, we found lower microglia/macrophage recruitment in RTRE in the recurrent tumors (p &lt; 0.01) and throughout the tumor areas compared to the RERT group. CONCLUSIONS: Our study represents the first preclinical study demonstrating the feasibility and longer overall survival using RTRE instead of RERT in vivo. Moreover, RTRE shows an important effect in the macrophages/microglia recruitment and in the immune profile. This suggests strong superiority for new clinical radiation strategies.

Electrical stimulation affects the differentiation of transplanted regionally specific human spinal neural progenitor cells (sNPCs) after chronic spinal cord injury

BackgroundThere are currently no effective clinical therapies to ameliorate the loss of function that occurs after spinal cord injury. Electrical stimulation of the rat spinal cord through the rat tail has previously been described by our laboratory. We propose combinatorial treatment with human induced pluripotent stem cell-derived spinal neural progenitor cells (sNPCs) along with tail nerve electrical stimulation (TANES). The purpose of this study was to examine the influence of TANES on the differentiation of sNPCs with the hypothesis that the addition of TANES would affect incorporation of sNPCs into the injured spinal cord, which is our ultimate goal.MethodsChronically injured athymic nude rats were allocated to one of three treatment groups: injury only, sNPC only, or sNPC + TANES. Rats were sacrificed at 16 weeks post-transplantation, and tissue was processed and analyzed utilizing standard histological and tissue clearing techniques. Functional testing was performed. All quantitative data were presented as mean ± standard error of the mean. Statistics were conducted using GraphPad Prism.ResultsWe found that sNPCs were multi-potent and retained the ability to differentiate into mainly neurons or oligodendrocytes after this transplantation paradigm. The addition of TANES resulted in more transplanted cells differentiating into oligodendrocytes compared with no TANES treatment, and more myelin was found. TANES not only promoted significantly higher numbers of sNPCs migrating away from the site of injection but also influenced long-distance axonal/dendritic projections especially in the rostral direction. Further, we observed localization of synaptophysin on SC121-positive cells, suggesting integration with host or surrounding neurons, and this finding was enhanced when TANES was applied. Also, rats that were transplanted with sNPCs in combination with TANES resulted in an increase in serotonergic fibers in the lumbar region. This suggests that TANES contributes to integration of sNPCs, as well as activity-dependent oligodendrocyte and myelin remodeling of the chronically injured spinal cord.ConclusionsTogether, the data suggest that the added electrical stimulation promoted cellular integration and influenced the fate of human induced pluripotent stem cell-derived sNPCs transplanted into the injured spinal cord.

Therapeutic potential of human induced pluripotent stem cell–derived cardiac tissue in an ischemic model with unloaded condition mimicking left ventricular assist device

ObjectiveThis study aimed to explore the therapeutic potential of human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) in the emerging approach of bridge to recovery for severe heart failure with ventricular assist devices. We used a rat model of heterotopic heart transplantation (HTx) to mimic ventricular assist device support and heart unloading. MethodsHiCTs were created by inserting gelatin hydrogel microspheres between cell sheets made from hiPSC-derived cardiovascular cells. Male athymic nude rats underwent myocardial infarction (MI) and were divided into the following groups: MI (loaded, untreated control), MI + HTx (unloaded, untreated control), MI + HTx + HiCT (unloaded, treated), and MI + HiCT (loaded, treated). HiCTs were placed on the epicardium of the heart in treated groups. We evaluated HiCT engraftment, fibrosis, and neovascularization using histologic analysis. ResultsAfter 4 weeks, HiCTs successfully engrafted in 5 of 6 rats in the MI + HTx + HiCT group (83.3%). The engrafted HiCT area was greater under unloaded conditions (MI + HTx + HiCT) than loaded conditions (MI + HiCT) (P < .05). MI + HTx + HiCT had a significantly smaller infarct area compared with MI and MI + HTx. The MI + HTx + MiCT group exhibited greater vascular density in the border zone than MI and MI + HTx. HiCT treatment suppressed cardiomyocyte atrophy due to left ventricular unloading (P = .001). The protein level of muscle-specific RING finger 1, an atrophy-related ubiquitin ligase, was lower in the MI + HTx + HiCT group than in MI + HTx (P = .036). ConclusionsTransplanting HiCTs into ischemic hearts under unloaded conditions promoted engraftment, neovascularization, attenuated infarct remodeling, and suppressed myocyte atrophy. These results suggest that HiCT treatment could contribute to future advancements in bridge to recovery.

Abstract P422: CD4+ T Cells From Preeclamptic Patients During Pregnancy Causes Cognitive Dysfunction Postpartum In A Pregnant Rat Model

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with impaired cognitive function and increased cerebral blood flow (CBF). Although we have previously shown adoptive transfer of placental CD4+ T cells from PE women into athymic nude pregnant rats causes HTN and circulating factors associated with PE, we do not know the role of CD4+ T cells in contributing to impaired cerebral blood flow and cognitive dysfunction in the postpartum period. Therefore, we hypothesize that adoptive transfer of placental CD4+ T cells from PE patients into pregnant athymic nude recipient rats will cause maternal hypertension and cognitive dysfunction and impaired CBF postpartum. Placental CD4+ T cells were isolated from normotensive (NP) and preeclamptic (PE) pregnant women at delivery. One million CD4+ T cells were injected interperitoneally (i.p.) into normal pregnant nude athymic rats on gestational day (GD) 12. Dams were allowed to deliver and aged 12-weeks after delivery. Blood pressure (MAP) was measured by carotid catheter and at 12-weeks postpartum CBF autoregulation was measured by laser Doppler flowmetry. The Barnes maze and the novel object recognition behavioral assays were used to assess cognitive function on gestational day (GD) 15-19 and 12-weeks postpartum. A student’s t-test was used for statistical analysis. MAP increased during pregnancy in PE recipient rats (115±2 mmHg, n=5) compared to NP recipient rats (99±3 mmHg, n=4, p&lt; 0.05). MAP significantly increased in the postpartum period in PE recipient rats (114±2 mmHg, n=5) compared to NP controls (98±2 mmHg, n=7, p&lt;0.05). PE recipient rats exhibited latency with errors navigating in the Barnes maze compared to NP controls during pregnancy and postpartum. Locomotor activity was decreased in PE recipient rats compared to NP controls during pregnancy (p&lt;0.05) and postpartum. PE recipient rats had increased CBF autoregulation compared to NP rat recipients in the postpartum period. Our findings indicate that rat recipients of CD4+ T cells from PE patients demonstrate maternal HTN and increased CBF and cognitive dysfunction in the postpartum period compared to recipients of NP CD4 + T cells. These data indicate long-term cognitive and cerebral dysfunction as a result of preeclampsia.

Human iPSC-derived neural progenitor cells secreting GDNF provide protection in rodent models of ALS and retinal degeneration.

Human induced pluripotent stem cells (iPSCs) are a renewable cell source that can be differentiated into neural progenitor cells (iNPCs) and transduced with glial cell line-derived neurotrophic factor (iNPC-GDNFs). The goal of the current study is to characterize iNPC-GDNFs and test their therapeutic potential and safety. Single-nuclei RNA-seq show iNPC-GDNFs express NPC markers. iNPC-GDNFs delivered into the subretinal space of the Royal College of Surgeons rodent model of retinal degeneration preserve photoreceptors and visual function. Additionally, iNPC-GDNF transplants in the spinal cord of SOD1G93A amyotrophic lateral sclerosis (ALS) rats preserve motor neurons. Finally, iNPC-GDNF transplants in the spinal cord of athymic nude rats survive and produce GDNF for 9months, with no signs of tumor formation or continual cell proliferation. iNPC-GDNFs survive long-term, are safe, and provide neuroprotection in models of both retinal degeneration and ALS, indicating their potential as a combined cell and gene therapy for various neurodegenerative diseases.

Establishment of a robust rat hepatitis E virus fecal-oral infection model and validation for antiviral studies

The hepatitis E virus (HEV) is a major cause of hepatitis, with an estimated 3.3 million symptomatic cases annually. There is no HEV-specific treatment besides the off-label use of ribavirin and a vaccine is only available in China and Pakistan. To aid the development of therapeutic and preventive strategies, there is a need for convenient HEV infection models in small laboratory animals. To this end, we make use of the rat hepatitis E virus. Human infections with this virus have been reported in recent years, making it a relevant pathogen for the establishment of a small animal infection model. We here report that oral gavage of a feces suspension, containing a pre-defined viral RNA load, results in a reproducible synchronized infection in athymic nude rats. This route of administration mimics fecal-oral transmission in a standardized fashion. The suitability of the model to study the effect of antiviral drugs was assessed by using ribavirin, which significantly reduced viral loads in the feces, liver, and other tissues.

Preliminary evaluation of the safety and efficacy of glucose solution infusion through the hepatic artery on irreversible electroporation focusing

Due to electrical features of the tissue, such as impedance, which have a significant impact on irreversible electroporation (IRE) function, the administration of glucose solution 5% (GS5%) through the hepatic artery would focus IRE on scattered liver tumors. By creating a differential impedance between healthy and tumor tissue. This study aimed to determine the effects of the GS5% protocol on healthy liver tissue and its safety. 21 male Athymic nude rats Hsd: RH-Foxn1mu were used in the study. Animals were split into two groups. In group 1, a continuous infusion through the gastroduodenal artery of GS5% was performed to measure the impedance with a dose of 0.008 mL/g for 16 min. In group 2, the animals were divided into two subgroups for infusions of GS5%. Group 2.1, at 0.008 mL/g for 16 min. Group 2.2 at 0.03 mL/g for 4 min. Blood samples were collected after anesthesia has been induced. The second sample, after catheterization of the artery, and the third after the GS5% infusion. All the animals were sacrificed to collect histological samples. The survival rate during the experiment was 100%. A considerable impact on the impedance of the tissue was noticed, on average up to 4.31 times more than the baseline, and no side effects were observed after GS5% infusion. In conclusion, impedance alteration by Glucose solution infusion may focus IRE on tumor tissue and decrease IRE’s effects on healthy tissue.

Adoptive transfer of CD4+ T cells from women with preeclampsia with or without a history of COVID during pregnancy contribute to hypertension, AT1-AA, and neurovascular dysfunction in pregnant rats

Objective: Preeclampsia (PE) is characterized by new onset hypertension during pregnancy and is associated with chronic inflammation and neurovascular dysfunction. The inflammation in PE includes activated CD4+ T cells and B cells producing agonistic antibodies against the angiotensin II type 1 receptor (AT1AA) which has also been shown to be produced in patients with COVID-19 (CV) infection. Interestingly, having had CV during pregnancy increases the incidence of a PE-like phenotype in that pregnancy. Both PE and CV have long lasting neurological implications and we have shown that AT1AA mediates neurovascular dysfunction in PE. Moreover, we have shown that CD4+ T cells from women with PE induce a PE syndrome in pregnant athymic nude recipient rats. Although CD4+T cells have been shown to contribute to neurovascular dysfunction, we do not know the role of CD4+ T cells in contributing to neurovascular dysfunction in PE or in patients with a history (Hx) CV during pregnancy. Therefore, we sought examine a role for CD4+ T cells from PE with or without a CV Hx to contribute to AT1AA in pregnancy as well as hypertension and cerebrovascular dysfunction postpartum. We utilized adoptive transfer of placental CD4+ T cells from PE and normotensive (NT) patients with or without a CV Hx during pregnancy into athymic nude recipient rats and determined the presence of a PE phenotype during pregnancy or postpartum. Methods: CD4+ T cells were isolated from placentas of normal pregnant (NP), PE, CV Hx normotensive (CV Hx NT), and Hx of CV with PE (CV Hx PE) at delivery. One million CD4+ T cells were injected i.p. into gestational day (GD) 12 nude athymic rats. At GD19 blood samples were collected to measure AT1AA by cardiomyocyte assay. At three months postpartum, MAP and CBF were measured by arterial catheter and laser Doppler flowmetry. A two-way ANOVA was used for statistical analysis. Results: In pregnant recipient rats, AT1AA was increased in PE (8±2 ΔBPM, n=4) and CV Hx PE (4±2 ΔBPM, n=4) compared to NP (-3±2 ΔBPM, n=4, p&lt;0.05) and CV Hx NT (2±2 ΔBPM, n=2) respectively. At 3 months postpartum, MAP was increased in PE (115±2 mmHg, n=5) and CV Hx PE (111±4, n=4) compared to NP (99±3 mmHg, n=4 p&lt;0.05) and CV Hx NT (100±4, n=5). PE and CV Hx PE had impaired autoregulation of CBF compared to CV Hx NT and NP rats. Conclusion: Our findings indicate that CD4+ T cells from PE or CV HX PE patients cause AT1-AA in pregnancy as well as hypertension and impaired CBF 3 months postpartum. Collectively, the data demonstrates that CD4+ T cells cause hypertension and inflammation in PE or CV Hx PE and may contribute to neurovascular dysfunction in patients with previous PE or CV Hx PE during pregnancy. RO1HD067541-06, P20GM121334, and AHA 19CDA34670055 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring the neuroprotective effects of montelukast on brain inflammation and metabolism in a rat model of quinolinic acid-induced striatal neurotoxicity

BackgroundOne intrastriatal administration of quinolinic acid (QA) in rats induces a lesion with features resembling those observed in Huntington’s disease. Our aim is to evaluate the effects of the cysteinyl leukotriene receptor antagonist montelukast (MLK), which exhibited neuroprotection in different preclinical models of neurodegeneration, on QA-induced neuroinflammation and regional metabolic functions.MethodsThe right and left striatum of Sprague Dawley and athymic nude rats were injected with QA and vehicle (VEH), respectively. Starting from the day before QA injection, animals were treated with 1 or 10 mg/kg of MLK or VEH for 14 days. At 14 and 30 days post-lesion, animals were monitored with magnetic resonance imaging (MRI) and positron emission tomography (PET) using [18F]-VC701, a translocator protein (TSPO)-specific radiotracer. Striatal neuroinflammatory response was measured post-mortem in rats treated with 1 mg/kg of MLK by immunofluorescence. Rats treated with 10 mg/kg of MLK also underwent a [18F]-FDG PET study at baseline and 4 months after lesion. [18F]-FDG PET data were then used to assess metabolic connectivity between brain regions by applying a covariance analysis method.ResultsMLK treatment was not able to reduce the QA-induced increase in striatal TSPO PET signal and MRI lesion volume, where we only detected a trend towards reduction in animals treated with 10 mg/kg of MLK. Post-mortem immunofluorescence analysis revealed that MLK attenuated the increase in striatal markers of astrogliosis and activated microglia in the lesioned hemisphere. We also found a significant increase in a marker of anti-inflammatory activity (MannR) and a trend towards reduction in a marker of pro-inflammatory activity (iNOS) in the lesioned striatum of MLK—compared to VEH-treated rats. [18F]-FDG uptake was significantly reduced in the striatum and ipsilesional cortical regions of VEH-treated rats at 4 months after lesion. MLK administration preserved glucose metabolism in these cortical regions, but not in the striatum. Finally, MLK was able to counteract changes in metabolic connectivity and measures of network topology induced by QA, in both lesioned and non-lesioned hemispheres.ConclusionsOverall, MLK treatment produced a significant neuroprotective effect by reducing neuroinflammation assessed by immunofluorescence and preserving regional brain metabolism and metabolic connectivity from QA-induced neurotoxicity in cortical and subcortical regions.

Engineered tissue vascularization and engraftment depends on host model

Developing vascular networks that integrate with the host circulation and support cells engrafted within engineered tissues remains a key challenge in tissue engineering. Most previous work in this field has focused on developing new methods to build human vascular networks within engineered tissues prior to their implant in vivo, with substantively less attention paid to the role of the host in tissue vascularization and engraftment. Here, we assessed the role that different host animal models and anatomic implant locations play in vascularization and cardiomyocyte survival within engineered tissues. We found major differences in the formation of graft-derived blood vessels and survival of cardiomyocytes after implantation of identical tissues in immunodeficient athymic nude mice versus rats. Athymic mice supported robust guided vascularization of human microvessels carrying host blood but relatively sparse cardiac grafts within engineered tissues, regardless of implant site. Conversely, athymic rats produced substantive inflammatory changes that degraded grafts (abdomen) or disrupted vascular patterning (heart). Despite disrupted vascular patterning, athymic rats supported > 3-fold larger human cardiomyocyte grafts compared to athymic mice. This work demonstrates the critical importance of the host for vascularization and engraftment of engineered tissues, which has broad translational implications across regenerative medicine.

Viable cryopreserved human bone graft exhibit superior osteogenic properties in mandibular lateral augmentation

Lack of bone volume to place dental implants is frequently a problem in the reconstruction of edentulous patients. Even though autografts are the gold standard for jaw regeneration, morbidity associated with the harvesting site stimulates the demand for other substitutes. The aim of this study is to characterize the incorporation and the osteogenic ability of a viable cryopreserved human bone graft (VC-HBG) in the mandibular augmentation in rats. Bone chips from fresh human vertebrae cadaveric donors were processed, cryoprotected and deep-frozen at − 80 °C maintaining its cell viability. A jaw augmentation model was used in 20 athymic nude rats allocated into 2 groups to either receive the VC-HBG or an acellular graft as control (A-HBG). The assessment of the grafts' incorporation was performed at 4 and 8 weeks by micro-CT, histomorphometry and immunohistochemistry. Bone volume gain was significantly higher for the VC-HBG group at both time points. At 4 weeks, the A-HBG group presented significantly higher mineral density, but at 8 weeks, the VC-HBG group showed significantly higher values than the A-HBG. There was no statistical difference between VC-HBG and A-HBG groups at 4-weeks for remaining graft particles, while at 8 weeks, the VC-HBG group showed significantly less graft remnants. Collagen I, osteopontin and tartrate-resistant acid phosphatase expression were significantly higher in the VC-HBG group at both time points, while osteocalcin expression was significantly higher in the VC-HBG group at 8-weeks compared to the A-HBG group. This experimental research demonstrated that the VC-HBG shows positive osteogenic properties, greater bone formation, higher rate of bone remodeling and a better overall incorporation in rats' mandibles compared to the A-HBG.

Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap.

A healthy lymphatic system is required to return excess interstitial fluid back to the venous circulation. However, up to 49% of breast cancer survivors eventually develop breast cancer-related lymphedema due to lymphatic injuries from lymph node dissections or biopsies performed to treat cancer. While early-stage lymphedema can be ameliorated by manual lymph drainage, no cure exists for late-stage lymphedema when lymph vessels become completely dysfunctional. A viable late-stage treatment is the autotransplantation of functional lymphatic vessels. Here we report on a novel engineered lymphatic flap that may eventually replace the skin flaps used in vascularized lymph vessel transfers. The engineered flap mimics the lymphatic and dermal compartments of the skin by guiding multi-layered tissue organization of mesenchymal stem cells and lymphatic endothelial cells with an aligned decellularized fibroblast matrix. The construct was tested in a novel bilayered wound healing model and implanted into athymic nude rats. The in vitro model demonstrated capillary invasion into the wound gaps and deposition of extracellular matrix fibers, which may guide anastomosis and vascular integration of the graft during wound healing. The construct successfully anastomosed in vivo, forming chimeric vessels of human and rat cells. Overall, our flap replacement has high potential for treating lymphedema.

CD4+ T-Cells cause cognitive dysfunction in preeclampsia or COVID-19 infection in a pregnant rat model

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with increased cerebral blood flow (CBF), impaired cognitive function, and memory loss. We have shown adoptive transfer of placental CD4+ T cells from PE women into athymic nude pregnant rats causes HTN and increased circulating factors associated with PE. COVID-19 (CV) during pregnancy is associated with increased diagnosis of PE. However, the role of CD4+ T cells stimulated in response to CV in contributing to the PE phenotype in patients with a Hx of CV during pregnancy is unknown.