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Inhibition of transforming growth factor-? attenuates brain injury and neurological deficits in a rat model of germinal matrix hemorrhage.

ABSTRACT: Transforming growth factor-? (TGF-?) overproduction and activation of the TGF-? pathway are associated with the development of brain injury following germinal matrix hemorrhage (GMH) in premature infants. We examined the effects of GMH on the level of TGF-?1 in a novel rat collagenase-induced GMH model and determined the effect of inhibition of the TGF receptor I.In total, 92 seven-day old (P7) rats were used. Time-dependent effects of GMH on the level of TGF-?1 and TGF receptor I were evaluated by Western blot. A TGF receptor I inhibitor (SD208) was administered daily for 3 days, starting either 1 hour or 3 days after GMH induction. The effects of GMH and SD208 on the TGF-? pathway were evaluated by Western blot at day 3. The effects of GMH and SD208 on cognitive and motor function were also assessed. The effects of TGF receptor I inhibition by SD208 on GMH-induced brain injury and underlying molecular pathways were investigated by Western blot, immunofluorescence, and morphology studies 24 days after GMH.GMH induced significant delay in development, caused impairment in both cognitive and motor functions, and resulted in brain atrophy in rat subjects. GMH also caused deposition of both vitronectin (an extracellular matrix protein) and glial fibrillary acidic protein in perilesion areas, associated with development of hydrocephalus. SD208 ameliorated GMH-induced developmental delay, improved cognitive and motor functions, and attenuated body weight loss. SD208 also decreased vitronectin and glial fibrillary acidic protein deposition and decreased GMH-induced brain injury.Increased level of TGF-?1 and activation of the TGF-? pathway associate with the development of brain injury after GMH. SD208 inhibits GMH-induced activation of the TGF-? pathway and leads to an improved developmental profile, partial recovery of cognitive and motor functions, and attenuation of GMH-induced brain atrophy and hydrocephalus.

SUBMITTER: Manaenko A

PROVIDER: S-EPMC3966308 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Inhibition of transforming growth factor-β attenuates brain injury and neurological deficits in a rat model of germinal matrix hemorrhage.

Manaenko Anatol A Lekic Tim T Barnhart Margaret M Hartman Richard R Zhang John H JH

Stroke 20140114 3

<h4>Background and purpose</h4>Transforming growth factor-β (TGF-β) overproduction and activation of the TGF-β pathway are associated with the development of brain injury following germinal matrix hemorrhage (GMH) in premature infants. We examined the effects of GMH on the level of TGF-β1 in a novel rat collagenase-induced GMH model and determined the effect of inhibition of the TGF receptor I.<h4>Methods</h4>In total, 92 seven-day old (P7) rats were used. Time-dependent effects of GMH on the le ...[more]

PMID: 24425124

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Project description:Germinal matrix hemorrhage (GMH) is a serious complication in extremely preterm infants associated with neurological deficits and mortality. The purpose of the present study was to develop and characterize a grade III and IV GMH model in postnatal day 5 (P5) rats, the equivalent of preterm human brain maturation. P5 Wistar rats were exposed to unilateral GMH through intracranial injection into the striatum close to the germinal matrix with 0.1, 0.2, or 0.3 U of collagenase VII. During 10 days following GMH induction, motor functions and body weight were assessed and brain tissue collected at P16. Animals were tested for anxiety, motor coordination and motor asymmetry on P22-26 and P36-40. Using immunohistochemical staining and neuropathological scoring we found that a collagenase dose of 0.3 U induced GMH. Neuropathological assessment revealed that the brain injury in the collagenase group was characterized by dilation of the ipsilateral ventricle combined with mild to severe cellular necrosis as well as mild to moderate atrophy at the levels of striatum and subcortical white matter, and to a lesser extent, hippocampus and cortex. Within 0.5 h post-collagenase injection there was clear bleeding at the site of injury, with progressive increase in iron and infiltration of neutrophils in the first 24 h, together with focal microglia activation. By P16, blood was no longer observed, although significant gray and white matter brain infarction persisted. Astrogliosis was also detected at this time-point. Animals exposed to GMH performed worse than controls in the negative geotaxis test and also opened their eyes with latency compared to control animals. At P40, GMH rats spent more time in the center of open field box and moved at higher speed compared to the controls, and continued to show ipsilateral injury in striatum and subcortical white matter. We have established a P5 rat model of collagenase-induced GMH for the study of preterm brain injury. Our results show that P5 rat pups exposed to GMH develop moderate brain injury affecting both gray and white matter associated with delayed eye opening and abnormal motor functions. These animals develop hyperactivity and show reduced anxiety in the juvenile stage.

Project description:Perforin-mediated cytotoxicity plays a crucial role in microbial defense, tumor surveillance, and primary autoimmune disorders. However, the contribution of the cytolytic protein perforin to ischemia-induced secondary tissue damage in the brain has not been fully investigated. Here, we examined the kinetics and subpopulations of perforin-positive cells and then evaluated the direct effects of perforin-mediated cytotoxicity on outcomes after ischemic stroke. Using flow cytometry, we showed that perforin+CD45+ immune cells could be detected at 12 h and that the percentage of these cells increased largely until on day 3 and then significantly declined on day 7. Surprisingly, the percentage of Perforin+CD45+ cells also unexpectedly increased from day 7 to day 14 after ischemic stroke in Perforin1-EGFP transgenic mice. Our results suggested that Perforin+CD45+ cells play vital roles in the ischemic brain at early and late stages and further suggested that Perforin+CD45+ cells are a heterogeneous population. Surprisingly, in addition to CD8+ T cells, NK cells, and NKT cells, central nervous system (CNS)-resident immune microglia, which are first triggered and activated within minutes after ischemic stroke in mice, also secreted perforin during ischemic brain injury. In our study, the percentage of perforin+ microglia increased from 12 h after ischemic stroke, increased largely until on day 3 after ischemic stroke, and then moderately declined from days 3 to 7. Intriguingly, the percentage of perforin+ microglia also dramatically increased from days 7 to 14 after ischemic stroke. Furthermore, compared with wild-type littermates, Perforin 1-/- mice exhibited significant increases in the cerebral infarct volume, neurological deficits, and neurogenesis and inhibition of neurotoxic astrogliosis. Interestingly, the number of CD45+CD3+ T cells was significantly decreased in Perforin 1-/- mice compared with their wild-type littermates, especially the number of γδ T cells. In addition, Perforin 1-/- mice had lower levels of IL-17 than their wild-type littermates. Our results identified a critical function of perforin-mediated neurotoxicity in the ischemic brain, suggesting that targeting perforin-mediated neurotoxicity in brain-resident microglia and invading perforin+CD45+ immune cells may be a potential strategy for the treatment of ischemic stroke.

Project description:BACKGROUND:Neuroinflammation plays an important role in pathogenesis of germinal matrix hemorrhage (GMH). Neurotrophin-4 (NT-4) is a member of the neurotrophin family and interacts with the tropomyosin receptor kinase B (TrkB). NT-4 has been shown to confer neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4-TrkB signaling, as well as its downstream signaling cascade phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/forkhead box protein O1 (FoxO1), following GMH in neonatal rats. METHODS:GMH was induced by intraparenchymal injection of bacterial collagenase (0.3?U) in P7 rat pups. A total of 163 pups were used in this study. Recombinant human NT-4 was administered intranasally at 1?h after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002, and FoxO1 activating CRISPR were administered intracerebroventricularly at 24?h prior to NT-4 treatment to investigate the underlying mechanism. Short-term and long-term neurobehavioral assessments, immunofluorescence staining, Nissl's staining, and Western blot were performed. RESULTS:Expression of phosphorylated TrkB increased after GMH, reaching the peak level at day 3 after hemorrhage. TrkB receptors were observed on neurons, microglia, and astrocytes. The administration of rh-NT-4 induced phosphorylation of TrkB, expression of PI3K, and phosphorylation of Akt. Meanwhile, it decreased FoxO1 and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. FoxO1 activating CRISPR increased the expression of IL-6, suggesting that FoxO1 might be a potential inducer of pro-inflammatory factors. These results suggested that PI3K/Akt/FoxO1 signaling may be the downstream pathway of activation of TrkB. The rat pups treated with rh-NT-4 performed better than vehicle-treated animals in both short-term and long-term behavioral tests. CONCLUSION:These data showed that rh-NT-4 reduced the expression levels of pro-inflammatory cytokines, improved neurological function, attenuated neuroinflammation, and thereby mitigated post-hemorrhagic hydrocephalus after GMH by TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic strategy to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

Dataset Information

Inhibition of transforming growth factor-? attenuates brain injury and neurological deficits in a rat model of germinal matrix hemorrhage.

Publications

Inhibition of transforming growth factor-β attenuates brain injury and neurological deficits in a rat model of germinal matrix hemorrhage.

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