Fingolimod confers neuroprotection through activation of Rac1 after experimental germinal matrix hemorrhage in rat pups

CD200 has been reported to be neuroprotective in neurodegenerative diseases. However, the potential protective effects of CD200 in germinal matrix hemorrhage (GMH) have not been investigated. We examined the anti-inflammatory mechanisms of CD200 after GMH. A total of 167 seven-day-old rat pups were used. The time-dependent effect of GMH on the levels of CD200 and CD200 Receptor 1 (CD200R1) was evaluated by western blot. CD200R1 was localized by immunohistochemistry. The short-term (24 h) and long-term (28 days) outcomes were evaluated after CD200 fusion protein (CD200Fc) treatment by neurobehavioral assessment. CD200 small interfering RNA (siRNA) and downstream of tyrosine kinase 1 (Dok1) siRNA were injected intracerebroventricularly. Western blot was employed to study the mechanisms of CD200 and CD200R1. GMH induced significant developmental delay and caused impairment in both cognitive and motor functions in rat pups. CD200Fc ameliorated GMH-induced damage. CD200Fc increased expression of Dok1 and decreased IL-1beta and TNF-alpha levels. CD200R1 siRNA and Dok1 siRNA abolished the beneficial effects of CD200Fc, as demonstrated by enhanced expression levels of IL-1beta and TNF-alpha. CD200Fc inhibited GMH-induced inflammation and this effect may be mediated by CD200R1/Dok1 pathway. Thus, CD200Fc may serve as a potential treatment to ameliorate brain injury for GMH patients.

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NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

10.21203/rs.2.18641/v1 ◽

2019 ◽

Author(s):

Tianyi Wang ◽

Junyi Zhang ◽

Peng Li ◽

Yan Ding ◽

Jiping Tang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Inflammatory Factors ◽

Neonatal Rats ◽

Germinal Matrix ◽

Germinal Matrix Hemorrhage ◽

Trkb Receptor ◽

Rat Pups ◽

Short And Long Term ◽

Pro Inflammatory Cytokines

Abstract 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 it interacts with the tyrosine kinase B (TrkB) receptor. It has been studied that NT-4 has neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4 and it’s high affinity receptor TrkB as well as its downstream mediator phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/Forkhead box protein O1 (FoxO1) following GMH in neonatal rats, with a specific focus on inflammation. Methods: GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. A total of 163 seven-day-old pups were used in this study. The recombinant human NT-4 was administered intranasally at 1 hour after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002 and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 hours prior to NT-4 treatment to investigate the potential mechanism. Short-and-long-term neurobehavior assessments, immunofluorescence staining, Nissl’s staining and Western blot were performed. Results：The expression of p-TrkB increased after GMH with a peak at day3. The TrkB receptor was expressed by neurons, microglia, and astrocytes. The administration of rh-NT-4 increased phosphorylation of TrkB, expression of PI3K, phosphorylation of Akt and decreased FoxO1, IL-1beta and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. The use of FoxO1 activation CRISPR increased the expression of IL-6, suggesting that FoxO1 might potentially induce pro-inflammatory factors. These results demonstrated that PI3K/Akt/FoxO1 may be the downstream pathway of TrkB phosphorylation. The rat pups treated with rh-NT-4 performed better than untreated animals both in short-and-long-term behavior test. Conclusion：These data showed that rh-NT-4 can reduce the expression of pro-inflammatory cytokines, improve neurological function, attenuate neuroinflammation and post-hemorrhagic hydrocephalus after GMH by promoting TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic target to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

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A Model of Germinal Matrix Hemorrhage in Preterm Rat Pups

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.535320 ◽

2020 ◽

Vol 14 ◽

Author(s):

Masako Jinnai ◽

Gabriella Koning ◽

Gagandeep Singh-Mallah ◽

Andrea Jonsdotter ◽

Anna-Lena Leverin ◽

...

Keyword(s):

Brain Injury ◽

White Matter ◽

Subcortical White Matter ◽

Brain Maturation ◽

Germinal Matrix ◽

Motor Functions ◽

Germinal Matrix Hemorrhage ◽

Rat Pups ◽

Extremely Preterm ◽

Extremely Preterm Infants

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.

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NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

Journal of Neuroinflammation ◽

10.1186/s12974-020-01835-z ◽

2020 ◽

Vol 17 (1) ◽

Cited By ~ 2

Author(s):

Tianyi Wang ◽

Junyi Zhang ◽

Peng Li ◽

Yan Ding ◽

Jiping Tang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Inflammatory Factors ◽

Neonatal Rats ◽

Short Term ◽

Germinal Matrix ◽

Expression Levels ◽

Germinal Matrix Hemorrhage ◽

Rat Pups ◽

Pro Inflammatory Cytokines

Abstract 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.

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NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

10.21203/rs.2.18641/v2 ◽

2020 ◽

Author(s):

Tianyi Wang ◽

Junyi Zhang ◽

Peng Li ◽

Yan Ding ◽

Jiping Tang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Inflammatory Factors ◽

Neonatal Rats ◽

Germinal Matrix ◽

Germinal Matrix Hemorrhage ◽

Trkb Receptor ◽

Rat Pups ◽

Short And Long Term ◽

Pro Inflammatory Cytokines

Abstract 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 it interacts with the tyrosine kinase B (TrkB) receptor. It has been studied that NT-4 has neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4 and it’s high affinity receptor TrkB as well as its downstream mediator phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/Forkhead box protein O1 (FoxO1) following GMH in neonatal rats, with a specific focus on inflammation. Methods: GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. A total of 163 seven-day-old pups were used in this study. The recombinant human NT-4 was administered intranasally at 1 hour after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002 and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 hours prior to NT-4 treatment to investigate the potential mechanism. Short-and-long-term neurobehavior assessments, immunofluorescence staining, Nissl’s staining and Western blot were performed. Results ： The expression of p-TrkB increased after GMH with a peak at day3. The TrkB receptor was expressed by neurons, microglia, and astrocytes. The administration of rh-NT-4 increased phosphorylation of TrkB, expression of PI3K, phosphorylation of Akt and decreased FoxO1, IL-1beta and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. The use of FoxO1 activation CRISPR increased the expression of IL-6, suggesting that FoxO1 might potentially induce pro-inflammatory factors. These results demonstrated that PI3K/Akt/FoxO1 may be the downstream pathway of TrkB phosphorylation. The rat pups treated with rh-NT-4 performed better than untreated animals both in short-and-long-term behavior test. Conclusion： These data showed that rh-NT-4 can reduce the expression of pro-inflammatory cytokines, improve neurological function, attenuate neuroinflammation and post-hemorrhagic hydrocephalus after GMH by promoting TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic target to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

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NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

10.21203/rs.2.18641/v3 ◽

2020 ◽

Author(s):

Tianyi Wang ◽

Junyi Zhang ◽

Peng Li ◽

Yan Ding ◽

Jiping Tang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Inflammatory Factors ◽

Neonatal Rats ◽

Germinal Matrix ◽

Germinal Matrix Hemorrhage ◽

Trkb Receptor ◽

Rat Pups ◽

Short And Long Term ◽

Pro Inflammatory Cytokines

Abstract 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 it interacts with the tyrosine kinase B (TrkB) receptor. It has been studied that NT-4 has neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4 and it’s high affinity receptor TrkB as well as its downstream mediator phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/Forkhead box protein O1 (FoxO1) following GMH in neonatal rats, with a specific focus on inflammation. Methods: GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. A total of 163 seven-day-old pups were used in this study. The recombinant human NT-4 was administered intranasally at 1 hour after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002 and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 hours prior to NT-4 treatment to investigate the potential mechanism. Short-and-long-term neurobehavior assessments, immunofluorescence staining, Nissl’s staining and Western blot were performed. Results ： The expression of p-TrkB increased after GMH with a peak at day3. The TrkB receptor was expressed by neurons, microglia, and astrocytes. The administration of rh-NT-4 increased phosphorylation of TrkB, expression of PI3K, phosphorylation of Akt and decreased FoxO1, IL-1beta and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. The use of FoxO1 activation CRISPR increased the expression of IL-6, suggesting that FoxO1 might potentially induce pro-inflammatory factors. These results demonstrated that PI3K/Akt/FoxO1 may be the downstream pathway of TrkB phosphorylation. The rat pups treated with rh-NT-4 performed better than untreated animals both in short-and-long-term behavior test. Conclusion： These data showed that rh-NT-4 can reduce the expression of pro-inflammatory cytokines, improve neurological function, attenuate neuroinflammation and post-hemorrhagic hydrocephalus after GMH by promoting TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic target to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

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Technical Nuances in Neuroendoscopic Lavage for Germinal Matrix Hemorrhage in Preterm Infants: Twenty Tips and Pearls after More than One Hundred Procedures

Pediatric Neurosurgery ◽

10.1159/000516183 ◽

2021 ◽

pp. 1-9

Author(s):

Jorge Tirado-Caballero ◽

Jorge Herreria-Franco ◽

Mónica Rivero-Garvía ◽

Gloria Moreno-Madueño ◽

Maria Jose Mayorga-Buiza ◽

...

Keyword(s):

Preterm Infants ◽

Cerebrospinal Fluid Leak ◽

Tips And Tricks ◽

Germinal Matrix ◽

Suitable Alternative ◽

Posthemorrhagic Hydrocephalus ◽

Mortality And Morbidity ◽

Germinal Matrix Hemorrhage ◽

Preoperative Stage ◽

Fluid Leak

Introduction: Posthemorrhagic hydrocephalus in preterm infants is a serious entity related to high mortality and morbidity. Neuroendoscopic lavage (NEL) is a suitable alternative for the management of this pathology. However, as with every endoscopic technique, it requires some experience and several cases to master. Methods: We present a descriptive study of some technical nuances, tips, and tricks that have been learned in the last 8 years with over a hundred NELs performed in preterm infants. These variations are classified into 3 categories according to their temporal relationship with the surgical procedure: preoperative stage, intraoperative stage, and postoperative stage. We include a brief description of each one and the reasons why they are included in our current clinical practice. Results: Twenty tips and pearls were described in detail and are reported here. Preoperative, intraoperative, and postoperative variations were exposed and related to the most frequent complications of this procedure: infection, cerebrospinal fluid leak, and rebleeding. Conclusions: NEL is a useful technique for the management of germinal matrix hemorrhage in preterm infants. These technical nuances have improved the results of our technique and helped us to prevent complications related to the procedure.

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