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Perinatal hypoxic-ischemic (H/I) brain injury remains a major cause of neurologic disability. Because we have previously demonstrated that this insult depletes cells from the subventricular zone (SVZ), the goal of the present investigation was to compare the relative vulnerability to H/I of neural stem cells versus progenitors. The dorsolateral SVZs of P6 rats were examined at 2 to 48 hours of recovery from H/I using hematoxylin and eosin, in situ end labeling (ISEL), terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL), electron microscopy, and immunofluorescence. Pyknotic nuclei and ISEL+ cells were observed by 4 hours of recovery, peaked at 12 hours, and persisted for at least 48 hours. Many active-caspase3+ cells were observed at 12 hours and they comprised one third of the total TUNEL+ population. Electron microscopy revealed that hybrid cell deaths predominated at 12 hours of recovery. Importantly, few dying cells were observed in the medial SVZ, where putative stem cells reside, and no nestin+ medial SVZ cells showed caspase-3 activation. By contrast, active-caspase-3+/PSA-NCAM+ progenitors were prominent in the lateral SVZ. These data demonstrate that early progenitors are vulnerable to H/I, whereas neural stem cells are resilient. The demise of these early progenitors may lead to the depletion of neuronal and late oligodendrocyte progenitors, contributing to cerebral dysgenesis after perinatal insults.

Excessive inflammation has been implicated in the progressive neurodegeneration that occurs in multiple neurological diseases, including cerebral ischemia, and elevated levels of the proinflammatory cytokine interleukin-1 (IL-1) have been shown to exacerbate brain damage, whereas diminishing IL-1 levels limits the extent of injury. However, to date there is no consensus regarding which receptor(s) mediates the detrimental effects of IL-1. Because we have previously demonstrated that signaling through the IL-1 type 1 receptor (IL-1R1) is necessary for microglial activation and because results from other studies have implicated microglia as effectors of neurodegeneration, we hypothesized that inactivating the IL-1R1 would decrease the extent of damage caused by a hypoxic-ischemic (H/I) insult. It is shown that a mild insult initiates progressive neurodegeneration that leads to cystic infarcts, which can be prevented by inactivating the IL-1R1. The IL-1R1 null mice also show preserved sensorimotor function at 1 month's recovery. The mild insult induces multiple proinflammatory cytokines and activates microglia, and these responses are dramatically curtailed in mice lacking the IL-1R1. Importantly, the neuroinflammation precedes the progressive enlargement of the infarct, suggesting that the inflammation is causal rather than a consequence of the brain damage. These findings show that abrogating the inflammation consequent to a mild H/I insult will prevent brain damage and preserve neurological function. Additionally, these data incriminate the IL-1R1 as a master proinflammatory cytokine receptor.

Cerebral hypoxia/ischemia of the newborn has a frequency of 4/1,000 births and remains a major cause of cerebral palsy, epilepsy, and mental retardation. Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, the data are incomplete regarding the mechanisms leading to permanent brain injury. Here we tested the hypothesis that cerebral hypoxia/ischemia damages stem/progenitor cells in the subventricular zone (SVZ), resulting in a permanent depletion of oligodendrocytes. We used a widely accepted rat model and examined animals at recovery intervals ranging from 4 h to 3 weeks. Within hours after the hypoxic-ischemic insult 20% of the total cells were deleted from the SVZ. The residual damaged cells appeared necrotic. During 48 h of recovery deaths accumulated; however, these later deaths were predominantly apoptotic. Many apoptotic SVZ cells stained with a marker for immature oligodendrocytes. At 3 weeks survival, the SVZ was smaller and markedly less cellular, and it contained less than 1/4 the normal complement of neural stem cells. The corresponding subcortical white matter was dysmyelinated, relatively devoid of oligodendrocytes and enriched in astrocytes. We conclude that neural stem cells and oligodendrocyte progenitors in the SVZ are vulnerable to hypoxia/ischemia. Consequently, the developmental production of oligodendrocytes is compromised and regeneration of damaged white matter oligodendrocytes does not occur resulting in failed regeneration of CNS myelin in periventricular loci. The resulting dysgenesis of the brain that occurs subsequent to perinatal hypoxic/ischemic injury may contribute to the cognitive and motor dysfunction that results from asphyxia of the newborn.

With significant improvements in neonatal care, fewer infants sustain severe injury as a consequence of hypoxia/ischemia (H/I). However, the majority of experimental studies have inflicted moderate to severe injuries, or they have assessed damage to the caudal forebrain; therefore, to better understand how a mild H/I episode affects the structures and cells of the rostral forebrain, we assessed the relative vulnerabilities of cells in the neocortex, striatum, corpus callosum, choroid plexus and subventricular zone (SVZ). To inflict mild H/I injury, the right common carotid artery was ligated followed by 1 h of hypoxia (8% O 2 ) at 37°C. Regional vulnerabilities were assessed using TUNEL, active caspase-3 and hematoxylin and eosin staining at 24 and 48 h of recovery. Scattered columns of cell death were observed in the neocortex with deep-layer neurons more vulnerable than more superficial neurons. The majority of these dying neurons appeared to be dying apoptotic rather than necrotic deaths. In addition, approximately 1/3 of the apoptotic cells in the neocortex were O4+ oligodendrocyte progenitors. We also observed a decrease in NG2 staining within the affected regions of the forebrain. By contrast, active caspase-3+/S-100β+ astrocytes were not observed. Neurons and O4+ oligodendrocyte progenitors also died apoptotic deaths within the striatum. The lining cells of the choroid plexus also sustained damage. Elevated numbers of apoptotic cells were observed in the most lateral region of the SVZ and some of these dying cells were O4+. The most novel finding of this study, that oligodendrocyte progenitors in the gray matter are damaged and eliminated as a consequence of perinatal H/I, provides new insights into the histopathology and neurological deficits observed in infants who sustain mild H/I brain injuries.

Hypoxia-ischemia (HI) is a leading cause of white matter damage, a major contributor to cerebral palsy in premature infants. Preferential white matter damage is believed to result from vulnerability of the immature oligodendrocyte (the pro-OL) to factors elevated during ischemic damage, such as oxygen free radicals and glutamate. In order to determine whether pro-OLs undergo apoptotic death after HI, we analyzed periventricular white matter OLs in P7 rats 4, 12 and 24 h after HI to analyze the time course and mode of cell death. DNA fragmentation was seen at 12 and 24 h of recovery after HI, representing a 17-fold increase over control. In addition, caspase-3 activation was found in NG2+ pro-OLs at 12 h. Electron-microscopic analysis of cell death in the white matter revealed a transition from early necrotic deaths to hybrid cell deaths to classical apoptosis between 4 and 24 h of recovery from HI. The delayed time course of apoptosis in pro-OLs supports the feasibility of interventions to improve clinical outcomes for newborns surviving birth asphyxia.

Hypoxia-ischemia (H/I) as a result of asphyxia at term remains a major cause of neurologic disability. Our previous studies in the P7 rat model of perinatal H/I have shown that progenitors within the subventricular zone (SVZ) are vulnerable to this insult. Since many investigators are using transgenic and knockout mice to determine the importance of specific molecules in the evolution of damage after a stroke, there is a need to perform comparative studies on the relative vulnerability of the mouse SVZ. Here we assess damage to the SVZ of 5-, 7- and 10-day-old C57BL/6 mice after unilateral common carotid artery cauterization followed by 70 min of H/I (10% O 2 ). Whereas 5- and 7-day-old mice sustained little SVZ damage as assessed by hematoxylin and eosin staining, there was a 16% reduction of cellularity in 10-day-old animals by 18 h of recovery. Additionally, swollen cells were observed in the medial region of the SVZ of 10-day-old mice. However, few caspase-3+ and TUNEL+ cells were observed in this region, which contains the putative neural stem cells. Rather, the majority of the dying cells were situated in the mediolateral and lateral tail of the SVZ. At 18 h of recovery, there was a 2-fold increase in the frequency of TUNEL+ cells in the ipsilateral SVZ as well as a 3-fold increase in the frequency of active-caspase-3+ cells. We conclude that progenitors within the neonatal mouse SVZ are vulnerable to hypoxic/ischemic insult. The demise of these early progenitors likely leads to depletion of neuronal and late oligodendrocyte progenitors, contributing to cerebral dysgenesis.

Cerebral hypoxia/ischemia (H/I) of the premature infant is a major cause of cerebral palsy and mental retardation. An important determinant of the ultimate outcome from this insult is the extent to which the stem cells and progenitors in the brain are affected. Irreversible injury to these cells will impair normal development of the infant’s brain and, hence, its function. In the present study, we examine early intervals after H/I to identify which cells in the periventricular region are most vulnerable. At 0 h of recovery from a perinatal H/I insult, the choroid plexus shows extensive necrotic damage. The adjacent ependymal and subependymal cells are also affected. Swelling of the ependymal and medial subependymal cells is observed; however, these cells rarely sustain permanent damage. By contrast, cells in the most lateral aspect of the subventricular zone (SVZ) show more delayed, but extensive apoptotic and hybrid cell deaths. Interestingly, activated macrophages/microglia are observed adjacent to the swollen ependymal cells as well as within the affected subependyma. We conclude that the choroid plexus is an especially vulnerable structure in the immature brain, whereas the ependymal and adjacent subependymal cells are relatively resistant to damage. As the medial aspect of the SVZ contains neural stem cells, we predict that neural stem cells will be especially resistant to perinatal H/I brain damage.

BackgroundMultiple clinical trials investigating the combination of immunotherapy (IO) with antibody drug conjugates (ADC), with or without concurrent chemotherapy, are underway and showing promising results. Mechanism-based triple combination therapies utilizing ADC, IO and a third agent offer an opportunity for additional patient benefit. We investigated the antitumor efficacy of the B7-H4 targeting topoisomerase ADC puxitatug samrotecan (Puxi-sam), the PD1/TIGIT targeting, Fc-reduced monovalent bispecific antibody rilvegostomig and the PARP1-selective inhibitor (PARP1i) saruparib in combination in experimental cancer models.MethodsA 3D tumor spheroid model was generated using RL95-2 endometrial cancer cells and tumor reactive T cells primed on HLA-A24 restricted survivin/BIRC5 tumor antigen peptides or RL95-2 whole cell lysates. Syngeneic MC38 tumor cell lines were engineered to stably express human B7-H4. Tumor cells were implanted in wild-type C57BL6 mice and when tumors reached between 100-200 mm3, mice were randomized to receive either monotherapy doublet or triple combination therapy. Puxi-sam was administered as a single i.v. dose at 7 mg/kg, saruparib was administered orally daily for 21 days at 0.1 mg/kg and the murine surrogate of rilvegostomig was administered twice weekly i.p. at 10 mg/kg. A subset of mice had tumors harvested at 11 days post onset of dosing for pharmacodynamic evaluation by flow cytometry.ResultsIn vitro treatment of endometrial spheroid-T cell co-cultures with ADC-IO, ADC-PARPi and triplet combination induced additive tumor spheroid growth inhibition. In the fully immunocompetent syngeneic mouse model, the combination of Puxi-sam and rilvegostomig induced significant tumor growth inhibition (p<0.0001) and improved survival compared to monotherapies. The addition of saruparib further enhanced the anti-tumor activity and resulted in a majority of animals having complete and durable tumor regression. A follow up pharmacodynamic study assessed a cohort of tumors collected 11 days post treatment initiation. Flow cytometry of the dissociated tumors revealed a greater than 2-fold increase in intratumoral CD3 positive T cells, including a greater than 3-fold increase in cytotoxic effector CD8 positive T cells after triplet therapy compared to untreated mouse tumors. Further, there were decreased intratumoral myeloid derived suppressor cells (MDSCs) and increased macrophages expressing the co-stimulatory molecule CD86 in the triplet therapy group compared to the untreated group.ConclusionsRobust anti-tumor activity and tumor immune microenvironment modulation can be achieved with the combination of patuxitug-samrotecan, saruparib and rilvegostomig.Ethics ApprovalThe studies described were approved by the Institutional Animal Care and Use Committee of Astrazeneca under protocol AUP22-25.

During development, the output of the subventricular zone (SZ) becomes increasingly restricted, yet it still harbors multipotential progenitors. The output of the SZ could be gated by selectively eliminating inappropriately specified progenitors. Using in situ end-labeling (ISEL) to identify apoptotic cells, nearly 60% of the ISEL + cells in the juvenile forebrain were localized to the SZ. Of these dying cells, at least 9% could be identified as neurons, 4% as astrocytes, and 12% as oligodendrocytes. The remainder were negative for the stem cell marker nestin, as well as other markers evaluated. To test the hypothesis that committed progenitors were under selective pressures, neural stem/progenitor cells were allowed to differentiate in vitro in the presence or absence of the caspase 3 inhibitor z-DEVD-fmk. DEVD increased neuronal production 10-fold over control cultures. By contrast, the development of astrocytes and oligodendrocytes was not affected. Altogether, these data support the hypothesis that selective forces within the postnatal rat forebrain control the types of precursors that emerge from the germinal matrix. Furthermore, they suggest that different mechanisms control neuronal versus glial cell numbers.

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.