Neuroprotective effects of VEGF in the enteric nervous system

Although the enteric nervous system (ENS) functions largely autonomously as part of the peripheral nervous system (PNS), it is connected to the central nervous system (CNS) via the gut–brain axis. In many neurodegenerative diseases, pathological changes occur in addition to gastrointestinal symptoms, such as alpha-synuclein aggregates in Parkinson’s disease, which are found early in the ENS. In both the CNS and PNS, vascular endothelial growth factor (VEGF) mediates neuroprotective and neuroregenerative effects. Since the ENS with its close connection to the microbiome and the immune system is discussed as the origin of neurodegenerative diseases, it is necessary to investigate the possibly positive effects of VEGF on enteric neurons. Using laser microdissection and subsequent quantitative RT-PCR as well as immunohistochemistry, for the first time we were able to detect and localize VEGF receptor expression in rat myenteric neurons of different ages. Furthermore, we demonstrate direct neuroprotective effects of VEGF in the ENS in cell cultures. Thus, our results suggest a promising approach regarding neuroprotection, as the use of VEGF (may) prevent neuronal damage in the ENS

Effects of progesterone on T-type-Ca2+^{2+}-channel expression in Purkinje cells

Plasticity of cerebellar Purkinje cells (PC) is influenced by progesterone via the classical progesterone receptors PR-A and PR-B by stimulating dendritogenesis, spinogenesis, and synaptogenesis in these cells. Dissociated PC cultures were used to analyze progesterone effects at a molecular level on the voltage-gated T-type-Ca$^{2+}$-channels Ca$_v$3.1, Ca$_v$3.2, and Ca$_v$3.3 as they helped determine neuronal plasticity by regulating Ca$^{2+}$-influx in neuronal cells. The results showed direct effects of progesterone on the mRNA expression of T-type-Ca$^{2+}$-channels, as well as on the protein kinases A and C being involved in downstream signaling pathways that play an important role in neuronal plasticity. For the mRNA expression studies of T-type-Ca$^{2+}$-channels and protein kinases of the signaling cascade, laser microdissection and purified PC cultures of different maturation stages were used. Immunohistochemical staining was also performed to characterize the localization of T-type-Ca$^{2+}$-channels in PC. Experimental progesterone treatment was performed on the purified PC culture for 24 and 48 hours. Our results show that progesterone increases the expression of Ca$_v$3.1 and Ca$_v$3.3 and associated protein kinases A and C in PC at the mRNA level within 48 hours after treatment at latest. These effects extend the current knowledge of the function of progesterone in the central nervous system and provide an explanatory approach for its influence on neuronal plasticity

Progesterone

The enteric nervous system (ENS) is an intrinsic network of neuronal ganglia in the intestinal tube with about 100 million neurons located in the myenteric plexus and submucosal plexus. These neurons being affected in neurodegenerative diseases, such as Parkinson’s disease, before pathological changes in the central nervous system (CNS) become detectable is currently a subject of discussion. Understanding how to protect these neurons is, therefore, particularly important. Since it has already been shown that the neurosteroid progesterone mediates neuroprotective effects in the CNS and PNS, it is now equally important to see whether progesterone has similar effects in the ENS. For this purpose, the RT-qPCR analyses of laser microdissected ENS neurons were performed, showing for the first time the expression of the different progesterone receptors (PR-A/B; mPRa, mPRb, PGRMC1) in rats at different developmental stages. This was also confirmed in ENS ganglia using immunofluorescence techniques and confocal laser scanning microscopy. To analyze the potential neuroprotective effects of progesterone in the ENS, we stressed dissociated ENS cells with rotenone to induce damage typical of Parkinson’s disease. The potential neuroprotective effects of progesterone were then analyzed in this system. Treatment of cultured ENS neurons with progesterone reduced cell death by 45%, underscoring the tremendous neuroprotective potential of progesterone in the ENS. The additional administration of the PGRMC1 antagonist AG205 abolished the observed effect, indicating the crucial role of PGRMC1 with regard to the neuroprotective effect of progesterone

Expression pattern of T-type Ca2+Ca^{2+} channels in cerebellar Purkinje cells after VEGF treatment

T-type $Ca^{2+}$ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type $Ca^{2+}$ channel isoforms, Cav3.1 (encoded by $\it Cacna1g$), Cav3.2 (encoded by $\it Cacna1h$), and Cav3.3 (encoded by $\it Cacna1i$), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type $Ca^{2+}$ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type $Ca^{2+}$ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity

Innovative in vivo\textit {in vivo} rat model for global cerebral hypoxia

$\bf Introduction:$ Severe acute global cerebral hypoxia can lead to significant disability in humans. Although different animal models have been described to study hypoxia, there is no endogenous model that considers hypoxia and its effect on the brain as an independent factor. Thus, we developed a minimally invasive rat model, which is based on the non-depolarizing muscle blocking agent rocuronium in anesthetized animals. This drug causes respiratory insufficiency by paralysis of the striated muscles. $\bf Methods:$ In this study, 14 rats underwent 12 min of hypoxemia with an oxygen saturation of approximately 60% measured by pulse oximetry; thereafter, animals obtained sugammadex to antagonize rocuronium immediately. $\bf Results:$ Compared to controls (14 rats, anesthesia only), hypoxic animals demonstrated significant morphological alterations in the hippocampus (cell decrease in the CA 1 region) and the cerebellum (Purkinje cell decrease), as well as significant changes in hypoxia markers in blood (Hif2$\alpha$, Il1$\beta$, Tgf1$\beta$, Tnf$\alpha$, S100b, cspg2, neuron-specific enolase), hippocampus (Il1$\beta$, Tnf$\alpha$, S100b, cspg2, NSE), and cerebellum (Hif1$\alpha$, Tnf$\alpha$, S100b, cspg2, NSE). Effects were more pronounced in females than in males. $\bf Discussion:$ Consequently, this model is suitable to induce hypoxemia with consecutive global cerebral hypoxia. As significant morphological and biochemical changes were proven, it can be used to investigate therapeutic and preventive drugs for global cerebral hypoxia

Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging

Bioluminescence imaging is a sensitive approach for longitudinal neuroimaging. Transgenic mice expressing luciferase under the promoter of doublecortin (DCX-luc), a specific marker of neuronal progenitor cells (NPC), allow monitoring of neurogenesis in living mice. Since the extent and time course of neurogenesis during autoimmune brain inflammation are controversial, we investigated neurogenesis in MOG-peptide induced experimental allergic encephalomyelitis (EAE) using DCX-luc reporter mice. We observed a marked, 2- to 4-fold increase of the bioluminescence signal intensity 10 days after EAE induction and a gradual decline 1–2 weeks thereafter. In contrast, immunostaining for DCX revealed no differences between EAE and control mice 2 and 4 weeks after immunization in zones of adult murine neurogenesis such as the dentate gyrus. $\textit {Ex vivo}$ bioluminescence imaging showed similar luciferase expression in brain homogenates of EAE and control animals. Apart from complete immunization including MOG-peptide also incomplete immunization with complete Freund´s adjuvant and pertussis toxin resulted in a rapid increase of the $\textit {in vivo}$ bioluminescence signal. Blood-brain barrier (BBB) leakage was demonstrated 10 days after both complete and incomplete immunization and might explain the increased bioluminescence signal $\textit {in vivo}$. We conclude, that acute autoimmune inflammation in EAE does not alter neurogenesis, at least at the stage of DCX-expressing NPC. Effects of immunization on the BBB integrity must be considered when luciferase is used as a reporter within the CNS during the active stage of EAE. Models with stable CNS-restricted luciferase expression could serve as technically convenient way to evaluate BBB integrity in a longitudinal manner

Fingolimod for irradiation-induced neurodegeneration

$\bf Background:$ Cranial irradiation is a common therapy for the treatment of brain tumors, but unfortunately patients suffer from side effects, particularly cognitive impairment, caused by neurodegenerative and neuroinflammatory mechanisms. Finding a therapeutic agent protecting hippocampal neurons would be beneficial. Fingolimod (FTY720), a sphingosine-1-phosphate receptor modulator approved for multiple sclerosis, is an immunosuppressant and known to enhance proliferation and differentiation of neuronal precursor cells (NPCs). $\bf Objectives:$ To investigate whether pre-treatment with FTY720 protects NPCs $\textit {in vitro}$ and $\textit {in vivo}$ from irradiation-induced damage. $\bf Methods:$ Neuronal precursor cells were isolated from E13 C57BL/6 wildtype mice, treated at day 0 of differentiation with FTY720 and irradiated on day 6 with 1 Gy. NPCs were analyzed for markers of cell death (PI, caspase-3), proliferation (Ki67), and differentiation (DCX, $\beta$III-tubulin). Adult C57BL/6 wildtype mice were treated with FTY720 (1 mg/kg) and received a single dose of 6 Gy cranial irradiation at day 7. Using immunohistochemistry, we analyzed DCX and BrdU as markers of neurogenesis and Iba1, GFAP, and CD3 to visualize inflammation in the dentate gyrus (DG) and the subventricular zone (SVZ). B6(Cg)-Tyrc-2J/J DCX-luc reporter mice were used for bioluminescence imaging to evaluate the effect of FTY720 on neurogenesis in the DG and the spinal cord of naïve mice. $\bf Results:$ FTY720 protected NPCs against irradiation induced cell death $\textit {in vitro}$. Treatment with FTY720 dose-dependently reduced the number of PI$^{+}$ cells 24 and 96 h after irradiation without effecting proliferation or neuronal differentiation. $\textit {In vivo}$ treatment resulted in a significant survival of DCX$^{+}$ neurons in the DG and the SVZ 4 weeks after irradiation as well as a slight increase of proliferating cells. FTY720 inhibited microglia activation 24 h after X-ray exposure in the DG, while astrocyte activation was unaffected and no lymphocyte infiltrations were found. In naïve mice, FTY720 treatment for 4 weeks had no effect on neurogenesis. $\bf Conclusion:$ FTY720 treatment of NPCs prior to X-ray exposure and of mice prior to cranial irradiation is neuroprotective. No effects on neurogenesis were found