A dual-omics approach on the effects of fibroblast growth factor-2 (FGF-2) on ventral tegmental area dopaminergic neurons in response to alcohol consumption in mice.

Harmful alcohol consumption is a major socioeconomic burden to the health system, as it can be the cause of mortality of heavy alcohol drinkers. The dopaminergic (DAergic) system is thought to play an important role in the pathogenesis of alcohol drinking behaviour; however, its exact role remains elusive. Fibroblast growth factor 2 (FGF-2), a neurotrophic factor, associated with both the DAergic system and alcohol consumption, may play an important role in DAergic neuroadaptations during alcohol abuse. Within this study, we aimed to clarify the role of endogenous FGF-2 on the DAergic system and whether there is a possible link to alcohol consumption. We found that lack of FGF-2 reduces the alcohol intake of mice. Transcriptome analysis of DAergic neurons revealed that FGF-2 knockout (FGF-2 KO) shifts the molecular fingerprint of midbrain dopaminergic (mDA) neurons to DA subtypes of the ventral tegmental area (VTA). In line with this, proteomic changes predominantly appear also in the VTA. Interestingly, these changes led to an altered regulation of the FGF-2 signalling cascades and DAergic pathways in a region-specific manner, which was only marginally affected by voluntary alcohol consumption. Thus, lack of FGF-2 not only affects the gene expression but also the proteome of specific brain regions of mDA neurons. Our study provides new insights into the neuroadaptations of the DAergic system during alcohol abuse and, therefore, comprises novel targets for future pharmacological interventions.

FGF2 activity regulates operant alcohol self-administration and mesolimbic dopamine transmission.

Fibroblast growth factor 2 (FGF2) is involved in the development and maintenance of the brain dopamine system. We previously showed that alcohol exposure alters the expression of FGF2 and its receptor, FGF receptor 1 (FGFR1) in mesolimbic and nigrostriatal brain regions, and that FGF2 is a positive regulator of alcohol drinking. Here, we determined the effects of FGF2 and of FGFR1 inhibition on alcohol consumption, seeking and relapse, using a rat operant self-administration paradigm. In addition, we characterized the effects of FGF2-FGFR1 activation and inhibition on mesolimbic and nigrostriatal dopamine neuron activation using in vivo electrophysiology. We found that recombinant FGF2 (rFGF2) increased the firing rate and burst firing activity of dopaminergic neurons in the mesolimbic and nigrostriatal systems and led to increased operant alcohol self-administration. In contrast, the FGFR1 inhibitor PD173074 suppressed the firing rate of these dopaminergic neurons, and reduced operant alcohol self-administration. Alcohol seeking behavior was not affected by PD173074, but this FGFR1 inhibitor reduced post-abstinence relapse to alcohol consumption, albeit only in male rats. The latter was paralleled by the increased potency and efficacy of PD173074 in inhibiting dopamine neuron firing. Together, our findings suggest that targeting the FGF2-FGFR1 pathway can reduce alcohol consumption, possibly via altering mesolimbic and nigrostriatal neuronal activity.

Chronic Voluntary Alcohol Consumption Alters Promoter Methylation and Expression of Fgf-2 and Fgfr1.

Alcohol abuse accounts for 3.3 million deaths annually, rendering it a global health issue. Recently, fibroblast growth factor 2 (FGF-2) and its target, fibroblast growth factor receptor 1 (FGFR1), were discovered to positively regulate alcohol-drinking behaviors in mice. We tested whether alcohol intake and withdrawal alter DNA methylation of Fgf-2 and Fgfr1 and if there is a correlation regarding mRNA expression of these genes. Blood and brain tissues of mice receiving alcohol intermittently over a six-week period were analyzed using direct bisulfite sequencing and qRT-PCR analysis. Assessment of Fgf-2 and Fgfr1 promoter methylation revealed changes in the methylation of cytosines in the alcohol group compared with the control group. Moreover, we showed that the altered cytosines coincided with binding motives of several transcription factors. We also found that Fgf-2 and Fgfr1 gene expression was significantly decreased in alcohol-receiving mice compared with control littermates, and that this effect was specifically detected in the dorsomedial striatum, a brain region involved in the circuitry of the reward system. Overall, our data showed alcohol-induced alterations in both mRNA expression and methylation pattern of Fgf-2 and Fgfr1. Furthermore, these alterations showed a reward system regional specificity, therefore, resembling potential targets for future pharmacological interventions.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

FGF2 is an endogenous regulator of alcohol reward and consumption.

Alcohol use disorder (AUD) is a chronic, relapsing disorder, characterized by escalating alcohol drinking and loss of control, with very limited available treatments. We recently reported that the expression of fibroblast growth factor 2 (Fgf2) is increased in the striatum of rodents following long-term excessive alcohol drinking and that the systemic or intra-striatal administration of recombinant FGF2 increases alcohol consumption. Here, we set out to determine whether the endogenous FGF2 plays a role in alcohol drinking and reward, by testing the behavioural phenotype of Fgf2 knockout mice. We found that Fgf2 deficiency resulted in decreased alcohol consumption when tested in two-bottle choice procedures with various alcohol concentrations. Importantly, these effects were specific for alcohol, as a natural reward (sucrose) or water consumption was not affected by Fgf2 deficiency. In addition, Fgf2 knockout mice failed to show alcohol-conditioned place preference (CPP) but showed normal fear conditioning, suggesting that deletion of the growth factor reduces alcohol's rewarding properties. Finally, Fgf2 knockout mice took longer to recover from the loss of righting reflex and showed higher blood alcohol concentrations when challenged with an intoxicating alcohol dose, suggesting that their ethanol metabolism might be affected. Together, our results show that the endogenous FGF2 plays a critical role in alcohol drinking and reward and indicate that FGF2 is a positive regulator of alcohol-drinking behaviours. Our findings suggest that FGF2 is a potential biomarker for problem alcohol drinking and is a potential target for pharmacotherapy development for AUD.

Neuropathic pain: Spotlighting anatomy, experimental models, mechanisms, and therapeutic aspects.

The International Association for the Study of Pain defines neuropathic pain as "pain arising as a direct consequence of a lesion or disease affecting the somatosensory system". The associated changes can be observed in the peripheral as well as the central nervous system. The available literature discusses a wide variety of causes as predisposing for the development and amplification of neuropathic pain. Further, key interactions within sensory pathways have been discovered, but no common molecular mechanism leading to neuropathic pain has been identified until now. In the first part of this review, the pain mediating lateral spinothalamic tract is described. Different in vivo models are presented that allow studying trauma-, chemotherapy-, virus-, and diabetes-induced neuropathic pain in rodents. We furthermore discuss approaches to assess neuropathic pain in these models. Second, the current knowledge about cellular and molecular mechanisms suggested to underlie the development of neuropathic pain is presented and discussed. A summary of established therapies that are already applied in the clinic and novel, promising approaches closes the paper. In conclusion, the established animal models are able to emulate the diversity of neuropathic pain observed in the clinics. However, the assessment of neuropathic pain in the presented in vivo models should be improved. The determination of common molecular markers with suitable in vitro models would simplify the assessment of neuropathic pain in vivo. This would furthermore provide insights into common molecular mechanisms of the disease and establish a basis to search for satisfying therapeutic approaches.

Modification of tubular chitosan-based peripheral nerve implants: applications for simple or more complex approaches.

Surgical treatment of peripheral nerve injuries is still a major challenge in human clinic. Up to now, none of the well-developed microsurgical treatment options is able to guarantee a complete restoration of nerve function. This restriction is also effective for novel clinically approved artificial nerve guides. In this review, we compare surgical repair techniques primarily for digital nerve injuries reported with relatively high prevalence to be valuable attempts in clinical digital nerve repair and point out their advantages and shortcomings. We furthermore discuss the use of artificial nerve grafts with a focus on chitosan-based nerve guides, for which our own studies contributed to their approval for clinical use. In the second part of this review, very recent future perspectives for the enhancement of tubular (commonly hollow) nerve guides are discussed in terms of their clinical translatability and ability to form three-dimensional constructs that biomimick the natural nerve structure. This includes materials that have already shown their beneficial potential in in vivo studies like fibrous intraluminal guidance structures, hydrogels, growth factors, and approaches of cell transplantation. Additionally, we highlight upcoming future perspectives comprising co-application of stem cell secretome. From our overview, we conclude that already simple attempts are highly effective to increase the regeneration supporting properties of nerve guides in experimental studies. But for bringing nerve repair with bioartificial nerve grafts to the next level, e.g. repair of defects > 3 cm in human patients, more complex intraluminal guidance structures such as innovatively manufactured hydrogels and likely supplementation of stem cells or their secretome for therapeutic purposes may represent promising future perspectives.

What Can We Learn from FGF-2 Isoform-Specific Mouse Mutants? Differential Insights into FGF-2 Physiology In Vivo.

Fibroblast growth factor 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell growth, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and 22 kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a 22, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.

FGF-2 isoforms influence the development of dopaminergic neurons in the murine substantia nigra, but not anxiety-like behavior, stress susceptibility, or locomotor behavior.

BACKGROUND: Loss of fibroblast growth factor 2 (FGF-2) is responsible for the development of an increased number of dopaminergic (DA) neurons in the murine substantia nigra pars compacta (SNpc). Furthermore, dysregulation of its expression patterns within the central nervous system (CNS) is associated with behavioral abnormalities in mice. Until now, the contributions of the individual FGF-2 isoforms (one low (LMW) and two high molecular weight (HMW) isoforms) in the CNS are elusive. METHODS: To unravel the specific effects of FGF-2 isoforms, we compared three knockout mouse lines, one only deficient for LMW, one deficient for HMW and another lacking both isoforms, regarding DA neuronal development. With this regard, three time points of ontogenic development of the SNpc were stereologically investigated. Furthermore, behavioral aspects were analyzed in young adult mice, supplemented by corticosterone measurements. RESULTS: Juvenile mice lacking either LMW or HMW develop equal supernumerary DA neuron numbers in the SNpc. Compensatory increased LMW expression is observed in animals lacking HMW. Meanwhile, no knockout mouse line demonstrated changes in anxiety-like behavior, stress susceptibility, or locomotor behavior. CONCLUSIONS: Both FGF-2 isoforms crucially influence DA neuronal development in the murine SNpc. However, absence of LMW or HMW alone alters neither anxiety-like nor locomotor behavior, or stress susceptibility. Therefore, FGF-2 is not a determinant and causative factor for behavioral alterations alone, but probably in combination with appropriate conditions, like environmental or genetic factors.

AAV2/DJ-mediated alpha-synuclein overexpression in the rat substantia nigra as early stage model of Parkinson's disease.

Parkinson's disease (PD) is pathologically characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and alpha-synucleinopathy. We mimic the disease pathology with overexpression of either the human α-syn wildtype (α-syn-WT) or E46K mutant form (α-syn-E46K) in DA neurons of the SNpc in adult rats using AAV2/DJ as a viral vector for the first time. Transduction efficiency was compared to an equal virus titer expressing the green fluorescent protein (GFP). Motor skills of all animals were evaluated in the cylinder and amphetamine-induced rotation test over a total time period of 12 weeks. Additionally, stereological quantification of DA cells and striatal fiber density measurements were performed every 4 weeks after injection. Rats overexpressing α-syn-WT showed a progressive loss of DA neurons with 40% reduction after 12 weeks accompanied by a greater loss of striatal DA fibers. In contrast, α-syn-E46K led to this reduction after 4 weeks without further progress. Insoluble α-syn positive cytoplasmic inclusions were observed in both groups within DA neurons of the SNpc and VTA. In addition, both α-syn groups developed a characteristic worsening of the rotational behavior over time. However, only the α-syn-WT group reached statistically significant different values in the cylinder test. Summarizing these effects, we established a motor symptom animal model of PD by using AAV2/DJ in the brain for the first time. Thereby, overexpressing of α-syn-E46K mimicked a rather pre-symptomatic stage of the disease, while the α-syn-WT overexpressing animals imitated an early symptomatic stage of PD.

Isoform-selective as opposed to complete depletion of fibroblast growth factor 2 (FGF-2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice.

We have previously shown that total knockout of fibroblast growth factor-2 (FGF-2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1G93A ) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1G93A mice, with distinct regulation patterns of FGF-2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF-2-isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1G93A FGF-2 high molecular weight- and SOD1G93A FGF-2 low molecular weight-knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform-dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1G93A FGF-2 low molecular weight knockout mice compared to single mutant SOD1G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1G93A FGF-2 low molecular weight knockout mice implies that FGF-2 low molecular weight knockout (or the presence of the FGF-2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro.

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.

Introduction: Thematic Papers Issue on Peripheral Nerve Regeneration and Repair.

Injuries to the peripheral nerves result in loss of motor, sensory and autonomic functions in the denervated segments of the body, thus having strong impact in the quality of life of affected patients. Neurons are able to regenerate their injured axons in the peripheral nerves; however, the endogenous repair mechanisms usually do not allow for a satisfactory functional recovery, especially after severe nerve injuries. The interest on regeneration after peripheral nerve injuries has increased in the recent years due to the numerous advances derived from studies of neurobiology, cell therapy, and tissue engineering. This Thematic Papers Issue brings together a number of papers, authored by researchers in the field, which cover a wide spectrum of topics related to regeneration and repair of peripheral nerve injuries. The Issue proposal originated from the recent 4th International Symposium on Peripheral Nerve Regeneration (ISPNR2017) which was hosted by Xavier Navarro and the European Society for the Study of Peripheral Nerve Repair and Regeneration in Barcelona, Spain. Anat Rec, 301:1614-1617, 2018. © 2018 Wiley Periodicals, Inc.

Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology.

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

Neuronal firing activity in the basal ganglia after striatal transplantation of dopamine neurons in hemiparkinsonian rats.

The loss of nigral dopaminergic neurons and the resulting dopamine (DA) depletion in the striatum (STR) lead to altered neuronal activity and enhanced beta activity in various regions of the basal ganglia (BG) motor loop in patients with Parkinson's disease and in rodents in the 6-hydroxydopamine (6-OHDA)-lesioned rat model. Intrastriatal DA graft implantation has been shown to re-innervate the host brain and restore DA input. Here, DA cell grafts were implanted into the STR of 6-OHDA-lesioned rats and the effect on neuronal activity under urethane anesthesia (1.4g/kg, injected intraperitoneally) was tested in the entopeduncular nucleus (EPN, the equivalent to the human globus pallidus internus), the output nucleus of the BG, and the globus pallidus (GP, the equivalent to the human globus pallidus externus), a key region in the indirect pathway. In animals, which were transplanted with cells derived from the ventral mesencephalon of embryonic day 12 rat embryos into the STR, the rotational behavior induced by DA agonists in 6-OHDA-lesioned rats was significantly improved. This was accompanied by alleviated EPN firing rate and reinstated patterns of neuronal activity in the GP and EPN. Analysis of oscillatory activity revealed enhanced beta activity in both regions, which was reduced after grafting. In summary these data indicate restoration of BG motor loop toward normal activity by DA graft integration.

Regeneration of long-distance peripheral nerve defects after delayed reconstruction in healthy and diabetic rats is supported by immunomodulatory chitosan nerve guides.

BACKGROUND: Delayed reconstruction of transection or laceration injuries of peripheral nerves is inflicted by a reduced regeneration capacity. Diabetic conditions, more frequently encountered in clinical practice, are known to further impair regeneration in peripheral nerves. Chitosan nerve guides (CNGs) have recently been introduced as a new generation of medical devices for immediate peripheral nerve reconstruction. Here, CNGs were used for 45 days delayed reconstruction of critical length 15 mm rat sciatic nerve defects in either healthy Wistar rats or diabetic Goto-Kakizaki rats; the latter resembling type 2 diabetes. In short and long-term investigations, we comprehensively analyzed the performance of one-chambered hollow CNGs (hCNGs) and two-chambered CNGs (CFeCNGs) in which a chitosan film has been longitudinally introduced. Additionally, we investigated in vitro the immunomodulatory effect provided by the chitosan film. RESULTS: Both types of nerve guides, i.e. hCNGs and CFeCNGs, enabled moderate morphological and functional nerve regeneration after reconstruction that was delayed for 45 days. These positive findings were detectable in generally healthy as well as in diabetic Goto-Kakizaki rats (for the latter only in short-term studies). The regenerative outcome did not reach the degree as recently demonstrated after immediate reconstruction using hCNGs and CFeCNGs. CFeCNG-treatment, however, enabled tissue regrowth in all animals (hCNGs: only in 80% of animals). CFeCNGs did further support with an increased vascularization of the regenerated tissue and an enhanced regrowth of motor axons. One mechanism by which the CFeCNGs potentially support successful regeneration is an immunomodulatory effect induced by the chitosan film itself. Our in vitro results suggest that the pro-regenerative effect of chitosan is related to the differentiation of chitosan-adherent monocytes into pro-healing M2 macrophages. CONCLUSIONS: No considerable differences appear for the delayed nerve regeneration process related to healthy and diabetic conditions. Currently available chitosan nerve grafts do not support delayed nerve regeneration to the same extent as they do after immediate nerve reconstruction. The immunomodulatory characteristics of the biomaterial may, however, be crucial for their regeneration supportive effects.

Altered somatosensory cortex neuronal activity in a rat model of Parkinson's disease and levodopa-induced dyskinesias.

Several findings support the concept that sensorimotor integration is disturbed in Parkinson's disease (PD) and in levodopa-induced dyskinesias. In this study, we explored the neuronal firing activity of excitatory pyramidal cells and inhibitory interneurons in the forelimb region of the primary somatosensory cortex (S1FL-Ctx), along with its interaction with oscillatory activity of the primary motor cortex (MCtx) in 6-hydroxydopamine lesioned hemiparkinsonian (HP) and levodopa-primed dyskinetic (HP-LID) rats as compared to controls under urethane (1.4g/kg, i.p.) anesthesia. Further, gene expression patterns of distinct markers for inhibitory GABAergic neurons were analyzed in both cortical regions. While firing frequency and burst activity of S1FL-Ctx inhibitory interneurons were reduced in HP and HP-LID rats, measures of irregularity were enhanced in pyramidal cells. Further, enhanced coherence of distinct frequency bands of the theta/alpha, high-beta, and gamma frequency, together with enhanced synchronization of putative pyramidal cells and interneurons with MCtx oscillatory activity were observed. While GABA level was similar, gene expression levels of interneuron and GABAergic markers in S1FL-Ctx and MCtx of HP-LID rats differed to some extent. Our study shows that in a rat model of PD with dyskinesias, neuronal activity in putative interneurons was reduced, which was accompanied by high beta and gamma coherence between S1FL-Ctx and MCtx, together with changes in gene expression, indicating maladaptive neuroplasticity after long term levodopa treatment.

Fibroblast growth factor 23 signaling in hippocampal cells: impact on neuronal morphology and synaptic density.

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease, FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced number of primary neurites combined with a reduced arborization, resulting in a less complex morphology of neurons treated with FGF23. Moreover, FGF23 enhances the synaptic density in a FGF-receptor (FGF-R) dependent manner. Finally, we addressed the corresponding signaling events downstream of FGF-R employing a combination of western blots and quantitative immunofluorescence. Interestingly, FGF23 induces phospholipase Cγ activity in primary hippocampal neurons. Co-application of soluble α-Klotho leads to activation of the Akt-pathway and modifies FGF23-impact on neuronal morphology and synaptic density. Compared with other FGFs, this alternative signaling pattern is a possible reason for differential effects of FGF23 on hippocampal neurons and may thereby contribute to learning and memory deficits in chronic kidney disease patients. In this study, we show that fibroblast growth factor 23 inhibits neuronal ramification and enhances the synaptic density in primary hippocampal cultures accompanied by phospholipase Cγ-activation. Co-application of the co-receptor α-Klotho leads to an Akt-activation and further modifies neuronal morphology and number of synapses. Those effects provide a mechanistic basis for memory deficits in patients suffering from chronic kidney disease (CKD) characterized by excessively elevated FGF23 levels as well as memory deficits.

Chitosan-film enhanced chitosan nerve guides for long-distance regeneration of peripheral nerves.

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15 mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction.