Publications by authors named "Praneeti Pathipati"

13 Publications

  • Page 1 of 1

Mesenchymal Stem Cell (MSC)-Derived Extracellular Vesicles Protect from Neonatal Stroke by Interacting with Microglial Cells.

Neurotherapeutics 2021 Jul 7. Epub 2021 Jul 7.

Department of Neurology, UCSF, 675 Nelson Rising Lane, San Francisco, CA, 94158-0663, USA.

Mesenchymal stem cell (MSC)-based therapies are beneficial in models of perinatal stroke and hypoxia-ischemia. Mounting evidence suggests that in adult injury models, including stroke, MSC-derived small extracellular vesicles (MSC-sEV) contribute to the neuroprotective and regenerative effects of MSCs. Herein, we examined if MSC-sEV protect neonatal brain from stroke and if this effect is mediated via communication with microglia. MSC-sEV derived from bone marrow MSCs were characterized by size distribution (NanoSight™) and identity (protein markers). Studies in microglial cells isolated from the injured or contralateral cortex of postnatal day 9 (P9) mice subjected to a 3-h middle cerebral artery occlusion (tMCAO) and cultured (in vitro) revealed that uptake of fluorescently labeled MSC-sEV was significantly greater by microglia from the injured cortex vs. contralateral cortex. The cell-type-specific spatiotemporal distribution of MSC-sEV was also determined in vivo after tMCAO at P9. MSC-sEV administered at reperfusion, either by intracerebroventricular (ICV) or by intranasal (IN) routes, accumulated in the hemisphere ipsilateral to the occlusion, with differing spatial distribution 2 h, 18 h, and 72 h regardless of the administration route. By 72 h, MSC-sEV in the IN group was predominantly observed in Iba1 cells with retracted processes and in GLUT1 blood vessels in ischemic-reperfused regions. MSC-sEV presence in Iba1 cells was sustained. MSC-sEV administration also significantly reduced injury volume 72 h after tMCAO in part via modulatory effects on microglial cells. Together, these data establish feasibility for MSC-sEV delivery to injured neonatal brain via a clinically relevant IN route, which affords protection during sub-acute injury phase.
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http://dx.doi.org/10.1007/s13311-021-01076-9DOI Listing
July 2021

Neonatal stroke enhances interaction of microglia-derived extracellular vesicles with microglial cells.

Neurobiol Dis 2021 Sep 19;157:105431. Epub 2021 Jun 19.

Department of Neurology, UCSF, San Francisco, CA, USA. Electronic address:

Microglial cells support brain homeostasis under physiological conditions and modulate brain injury in a context-dependent and brain maturation-dependent manner. Microglial cells protect neonatal brain from acute stroke. While microglial signaling via direct cell-cell interaction and release of variety of molecules is intensely studied, less is known about microglial signaling via release and uptake of extracellular vesicles (EVs). We asked whether neonatal stroke alters release of microglial EVs (MEV) and MEV communication with activated microglia. We pulled down and plated microglia from ischemic-reperfused and contralateral cortex 24 h after transient middle cerebral artery occlusion (tMCAO) in postnatal day 9 mice, isolated and characterized microglia-derived microvesicles (P3-MEV) and exosomes (P4-MEV), and determined uptake of fluorescently labeled P3-MEV and P4-MEV by plated microglia derived from ischemic-reperfused and contralateral cortex. We then examined how reducing EVs release in neonatal brain-by intra-cortical injection of CRISPR-Cas9-Smpd3/KO (Smpd3/KD) to downregulate Smpd3 gene to disrupt neutral sphingomyelinase-2 (N-SMase2)-impacts P3-MEV and P4-MEV release and stroke injury. Both size and protein composition differed between P3-MEV and P4-MEV. tMCAO further altered protein composition of P3-MEV and P4-MEV and significantly, up to 5-fold, increased uptake of both vesicle subtypes by microglia from ischemic-reperfused regions. Under physiological conditions neurons were the predominant cell type expressing N-SMase-2, an enzyme involved in lipid signaling and EVs release. After tMCAO N-SMase-2 expression was diminished in injured neurons but increased in activated microglia/macrophages, leading to overall reduced N-SMase-2 activity. Compared to intracerebral injection of control plasmid, CRISPR-Cas9-Smpd3/Ct, Smpd3/KD injection further reduced N-SMase-2 activity and significantly reduced injury. Smpd3 downregulation decreased MEV release from injured regions, reduced Smpd3/KD-P3-MEV uptake and abolished Smpd3/KD-P4-MEV uptake by microglia from ischemic-reperfused region. Cumulatively, these data demonstrate that microglial cells release both microvesicles and exosomes in naïve neonatal brain, that the state of microglial activation determines both properties of released EVs and their recognition/uptake by microglia in ischemic-reperfused and control regions, suggesting a modulatory role of MEV in neonatal stroke, and that sphingosine/N-SMase-2 signaling contributes both to EVs release and uptake (predominantly P4-MEV) after neonatal stroke.
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http://dx.doi.org/10.1016/j.nbd.2021.105431DOI Listing
September 2021

Cryoprecipitate attenuates the endotheliopathy of trauma in mice subjected to hemorrhagic shock and trauma.

J Trauma Acute Care Surg 2021 06;90(6):1022-1031

From the Department of Surgery (M.B., D.S.) and Department of Laboratory Medicine (A.T., B.Y.M., L.R.V., M.K., H.Z., P.P., S.P.), University of California, San Francisco; Cerus Corporation (A.B., M.G.G.), Concord, California; and Shock Trauma Center (R.K.), University of Maryland School of Medicine, Baltimore, Maryland.

Background: Plasma has been shown to mitigate the endotheliopathy of trauma. Protection of the endothelium may be due in part to fibrinogen and other plasma-derived proteins found in cryoprecipitate; however, the exact mechanisms remain unknown. Clinical trials are underway investigating early cryoprecipitate administration in trauma. In this study, we hypothesize that cryoprecipitate will inhibit endothelial cell (EC) permeability in vitro and will replicate the ability of plasma to attenuate pulmonary vascular permeability and inflammation induced by hemorrhagic shock and trauma (HS/T) in mice.

Methods: In vitro, barrier permeability of ECs subjected to thrombin challenge was measured by transendothelial electrical resistance. In vivo, using an established mouse model of HS/T, we compared pulmonary vascular permeability among mice resuscitated with (1) lactated Ringer's solution (LR), (2) fresh frozen plasma (FFP), or (3) cryoprecipitate. Lung tissue from the mice in all groups was analyzed for markers of vascular integrity, inflammation, and inflammatory gene expression via NanoString messenger RNA quantification.

Results: Cryoprecipitate attenuates EC permeability and EC junctional compromise induced by thrombin in vitro in a dose-dependent fashion. In vivo, resuscitation of HS/T mice with either FFP or cryoprecipitate attenuates pulmonary vascular permeability (sham, 297 ± 155; LR, 848 ± 331; FFP, 379 ± 275; cryoprecipitate, 405 ± 207; p < 0.01, sham vs. LR; p < 0.01, LR vs. FFP; and p < 0.05, LR vs. cryoprecipitate). Lungs from cryoprecipitate- and FFP-treated mice demonstrate decreased lung injury, decreased infiltration of neutrophils and activation of macrophages, and preserved pericyte-endothelial interaction compared with LR-treated mice. Gene analysis of lung tissue from cryoprecipitate- and FFP-treated mice demonstrates decreased inflammatory gene expression, in particular, IL-1β and NLRP3, compared with LR-treated mice.

Conclusion: Our data suggest that cryoprecipitate attenuates the endotheliopathy of trauma in HS/T similar to FFP. Further investigation is warranted on active components and their mechanisms of action.
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http://dx.doi.org/10.1097/TA.0000000000003164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141010PMC
June 2021

Enhanced Mesenchymal Stromal Cells or Erythropoietin Provide Long-Term Functional Benefit After Neonatal Stroke.

Stroke 2021 01 22;52(1):284-293. Epub 2020 Dec 22.

Department of Pediatrics (A.L., P.P., S.O., A.R., D.F., F.F.G.), University of California, San Francisco.

Background And Purpose: Perinatal stroke is a common cause of life-long neurobehavioral compromise. Mesenchymal stromal cells (MSCs) and EPO (erythropoietin) have each demonstrated short-term benefit with delayed administration after stroke, and combination therapy may provide the most benefit. The purpose of this study is to determine the long-term histological and functional efficacy of enhanced, intranasal stem cell therapy (MSC preexposed to EPO) compared with standard MSC or multidose systemic EPO.

Methods: Transient middle cerebral artery occlusion or sham surgery was performed in postnatal day (P) 10 Sprague-Dawley rats, who were treated with single-dose intranasal MSC, MSC preexposed to EPO (MSC/EPO), multidose systemic EPO (EPO3; 1000 u/kg per dose×3 every 72 hours), or cell-conditioned media on P13 (day 3 [P13-P19] for EPO), or on P17 (day 7 [P17-P23] for EPO). At 2 months of age, animals underwent novel object recognition, cylinder rearing, and open field testing to assess recognition memory, sensorimotor function, and anxiety in adulthood.

Results: MSC, MSC/EPO, and EPO3 improved brain volume when administered at 3 or 7 days after middle cerebral artery occlusion. MSC/EPO also enhanced long-term recognition memory with either day 3 or day 7 treatment, but EPO3 had the most long-term benefit, improving recognition memory and exploratory behavior and reducing anxiety.

Conclusions: These data suggest that single-dose MSC/EPO and multidose systemic EPO improve long-term neurobehavioral outcomes even when administration is delayed, although EPO was the most effective treatment overall. It is possible that EPO represents a final common pathway for improved long-term repair, although the specific mechanisms remain to be determined.
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http://dx.doi.org/10.1161/STROKEAHA.120.031191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770074PMC
January 2021

Changes in arginase isoforms in a murine model of neonatal brain hypoxia-ischemia.

Pediatr Res 2021 03 28;89(4):830-837. Epub 2020 May 28.

Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.

Background: Arginases (ARG isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair; however, their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.

Methods: C57BL/6 mice subjected to the Vannucci procedure on postnatal day (P9) were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.

Results: ARG isoform expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 days. ARG activity during neurodevelopment remained unchanged, but increased at 1 day with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.

Conclusions: ARG isoform expression increases with age from P9 to P17, but is suppressed by injury specifically in the hippocampus and not in the cortex. Both levels and activity of ARG isoforms increase with H, while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG isoforms in the brain differ in spatiotemporal localization, expression, and activity during neurodevelopment and after injury.

Impact: Arginase isoforms change during neurodevelopment and after neonatal brain HI. This is the first study investigating the key enzymes of inflammation and tissue repair called arginases following murine neonatal brain HI. The highly region- and cell-specific expression suggests the possibility of specific functions of arginases. ARG-1 in microglia at the injury site may regulate neuroinflammation, while ARG-2 in neurons of developmental structures may impact neurodevelopment. While further studies are needed to describe the exact role of ARGs after neonatal brain HI, our study adds valuable data on anatomical localization and expression of ARGs in brain during development and after stroke.
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http://dx.doi.org/10.1038/s41390-020-0978-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704631PMC
March 2021

The Arginase Pathway in Neonatal Brain Hypoxia-Ischemia.

Dev Neurosci 2018 17;40(5-6):437-450. Epub 2019 Apr 17.

Department of Pediatrics, University of California San Francisco, San Francisco, California, USA.

Brain damage after hypoxia-ischemia (HI) occurs in an age-dependent manner. Neuroprotective strategies assumed to be effective in adults might have deleterious effects in the immature brain. In order to create effective therapies, the complex pathophysiology of HI in the developing brain requires exploring new mechanisms. Critical determinants of neuronal survival after HI are the extent of vascular dysfunction, inflammation, and oxidative stress, followed later by tissue repair. The key enzyme of these processes in the human body is arginase (ARG) that acts via the bioavailability of nitric oxide, and the synthesis of polyamines and proline. ARG is expressed throughout the brain in different cells. However, little is known about the effect of ARG in pathophysiological states of the brain, especially hypoxia-ischemia. Here, we summarize the role of ARG during neurodevelopment as well as in various brain pathologies.
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http://dx.doi.org/10.1159/000496467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784534PMC
April 2019

The Differential Effects of Erythropoietin Exposure to Oxidative Stress on Microglia and Astrocytes in vitro.

Dev Neurosci 2017 17;39(1-4):310-322. Epub 2017 May 17.

Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.

The neonatal brain is especially susceptible to oxidative stress owing to its reduced antioxidant capacity. Following hypoxic-ischemic (HI) injury, for example, there is a prolonged elevation in levels of hydrogen peroxide (H2O2) in the immature brain compared to the adult brain, resulting in lasting injury that can lead to life-long disability or morbidity. Erythropoietin (Epo) is one of few multifaceted treatment options that have been promising enough to trial in the clinic for both term and preterm brain injury. Despite this, there is a lack of clear understanding of how Epo modulates glial cell activity following oxidative injury, specifically, whether it affects microglia (Mg) and astrocytes (Ast) differently. Using an in vitro approach using primary murine Mg and Ast subjected to H2O2 injury, we studied the oxidative and inflammatory responses of Mg and Ast to recombinant murine (rm)Epo treatment. We found that Epo protects Ast from H2O2 injury (p < 0.05) and increases secreted nitric oxide levels in these cells (p < 0.05) while suppressing intracellular reactive oxygen species (p < 0.05) and superoxide ion (p < 0.05) levels only in Mg. Using a multiplex analysis, we noted that although H2O2 induced the levels of several chemokines, rmEpo did not have any significant specific effects on their levels, either with or without the presence of conditioned medium from injured neurons (NCM). Ultimately, it appears that rmEpo has pleiotropic effects based on the cell type; it has a protective effect on Ast but an antioxidative effect only on Mg without any significant modulation of chemokine and cytokine levels in either cell type. These findings highlight the importance of considering all cell types when assessing the benefits and pitfalls of Epo use.
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http://dx.doi.org/10.1159/000467391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961733PMC
February 2018

Phenotype and secretory responses to oxidative stress in microglia.

Dev Neurosci 2013 16;35(2-3):241-54. Epub 2013 Mar 16.

Department of Neurology, University of California San Francisco, San Francisco, CA 94158, USA.

The neonatal brain is particularly susceptible to oxidative stress. Our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (H2O2) levels rise significantly particularly in the neonatal brain and are sustained for up to 7 days. This rapidly accumulated H2O2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. To date, there is limited literature on the effects of increased H2O2 levels on microglial cells, which have been extensively implicated in the ensuing inflammatory injury. Microglial cultures were derived from the P1 mouse brain and exposed to either bolus concentrations of H2O2 (15 or 50 μM) or varying concentrations of continuous exposure for 4, 18 or 24 h. Continuous exposure of microglia to H2O2 was generated using the glucose oxidase-catalase system generating levels of H2O2 <10 μM. Reactive oxygen species and nitric oxide expression were measured. Conditioned medium was collected and analyzed for secreted cytokine levels. Treated cell extracts were processed for glutathione (oxidized and reduced) content and fixed cells were labeled for M1 and M2a phenotype markers. Overall, it is evident that microglial exposure to continuous H2O2 has pleiotropic and biphasic effects. Continuous exposure to very low levels of H2O2 is more damaging to cell survival than higher bolus doses at 18 h, and can produce considerably high levels of pro- and anti-inflammatory cytokines by 18 h. Significantly high levels of various chemokines/chemotactic molecules such as G-CSF, MIP-1b and MIP-2 are also produced in response to continuous low-dose H2O2 by 18 h. Interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. H2O2 exposure promotes an M2a microglial phenotype in the absence of IL-4/IL-13 signaling, suggesting a wound-healing role for microglia and a delayed activation mechanism for H2O2 after such an insult. Together, these specific effects can be used to clarify the microglial cell responses following injury in the immature brain.
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http://dx.doi.org/10.1159/000346159DOI Listing
March 2014

Developmental localization of NMDA receptors, Src and MAP kinases in mouse brain.

Neurosci Lett 2011 Oct 27;503(3):215-9. Epub 2011 Aug 27.

Department of Neurology, University of California, San Francisco, CA 94143, United States.

Activation of NMDA receptors (NMDAR) is associated with divergent downstream signaling leading to neuronal survival or death that may be regulated in part by whether the receptor is located synaptically or extrasynaptically. Distinct activation of the MAP kinases ERK and p38 by synaptic and extrasynaptic NMDAR is one of the mechanisms underlying these differences. We have recently shown that the Src family kinases (SFKs) play an important role in neonatal hypoxic-ischemic brain injury by regulating NMDAR phosphorylation. In this study, we characterized the distribution of NMDAR, SFKs and MAP kinases in synaptic and extrasynaptic membrane locations in the postnatal day 7 and adult mouse cortex. We found that the NMDAR, SFKs and phospho-NR2B were predominantly at synapses, whereas striatal-enriched protein tyrosine phosphatase (STEP) and its substrates ERK and p38 were much more concentrated extrasynaptically. NR1/NR2B was the main subunit at extrasynaptic membrane with concomitant NR2B phosphorylation at tyrosine (Y) 1336 in the immature brain. STEP expression increased, while p38 decreased with development in the extrasynaptic membrane. These results suggest that SFKs and STEP are poised to differentially regulate NMDAR-mediated signaling pathways due to their distinct subcellular localization, and thus may contribute to the age-specific differences seen in vulnerability, pathology and consequences of hypoxic-ischemic brain injury.
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http://dx.doi.org/10.1016/j.neulet.2011.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193348PMC
October 2011

Delayed and chronic treatment with growth hormone after endothelin-induced stroke in the adult rat.

Behav Brain Res 2009 Dec 27;204(1):93-101. Epub 2009 May 27.

Liggins Institute, University of Auckland, 2-6 Park Avenue, Grafton, Auckland, New Zealand.

We investigated the effects of a neurorestorative treatment paradigm using long-term, central delivery of growth hormone (GH) starting 4 days after stroke. It has been shown previously that a neural GH axis is activated after stroke, that GH is neuroprotective, and can have direct trophic actions on neurons and stem cells. First, we developed and validated a buffer that kept rat GH bioactive for 2 weeks at body temperature. Implanted minipumps were used to chronically infuse GH into the lateral ventricle of unilateral stroke injured adult rats. Initially, a dose ranging pilot study was used to characterize the neuroendocrine effects and distribution of the infused GH. Next, a 6-week treatment trial starting 4 days after induction of the stroke was performed and the animals allowed to recover for a further 6 weeks. Behavioural and endocrinological measures were taken. We found that the infused GH localized to cells within the ipsilateral; subventricular zone, white matter tract, lesion and penumbral regions. GH treatment accelerated recovery of one out of three tests of motor function (P<0.001) and improved spatial memory on the Morris water maze test at the end of the study (P<0.05), with no effect on learning. We also found that GH treatment was associated with a reversible increase in body weight (P<0.01) whilst circulating IGF-1 (insulin-like growth factor 1) levels were halved (P<0.001). Delayed and chronic treatment of stroke with central GH may accelerate some aspects of functional recovery and improve spatial memory in the long-term.
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http://dx.doi.org/10.1016/j.bbr.2009.05.023DOI Listing
December 2009

Selective losses of brainstem catecholamine neurons after hypoxia-ischemia in the immature rat pup.

Pediatr Res 2008 Apr;63(4):364-9

Perinatal Research Centre, University of Queensland, Queensland 4029, Australia.

Hypoxic-ischemic (HI) injury in the preterm neonate incurs numerous functional deficits, however little is known about the neurochemically-defined brain nuclei that may underpin them. Key candidates are the brainstem catecholamine neurons. Using an immature animal model, the postnatal day (P)-3 (P3) rat pup, we investigated the effects of HI on brainstem catecholamine neurons in the locus coeruleus, nucleus tractus solitarius (NTS), and ventrolateral medulla (VLM). On P21, we found that prior P3 HI significantly reduced numbers of catecholaminergic neurons in the locus coeruleus, NTS, and VLM. Only locus coeruleus A6, NTS A2, and VLM A1 noradrenergic neurons, but not NTS C2 and VLM C1 adrenergic neurons, were lost. There was also an associated reduction in dopamine-beta-hydroxylase-positive immunolabeling in the forebrain. These findings suggest neonatal HI can affect specific neurochemically-defined neuronal populations in the brainstem and that noradrenergic neurons are particularly vulnerable to HI injury.
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http://dx.doi.org/10.1203/PDR.0b013e3181659774DOI Listing
April 2008

Histopathological changes in insulin, glucagon and somatostatin cells in the islets of NOD mice during cyclophosphamide-accelerated diabetes: a combined immunohistochemical and histochemical study.

J Mol Histol 2005 May;36(4):289-300

School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

The cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.
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http://dx.doi.org/10.1007/s10735-005-7330-4DOI Listing
May 2005

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes.

Histochem Cell Biol 2003 Jun 11;119(6):451-61. Epub 2003 Jun 11.

School of Biological Sciences and the Department of Paediatrics, University of Auckland, Private Bag 92019, Auckland, New Zealand.

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1beta-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.
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http://dx.doi.org/10.1007/s00418-003-0537-0DOI Listing
June 2003
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