Basic fibroblast growth factor (bFGF) in rodent testis

We have previously described a 30 kDa basic fibroblast growth factor (bFGF)-like protein in rodent testicular homogenates and have shown that pachytene spermatocytes are the sites of predominant immunoreactivity for this bFGF-like protein (Mayerhofer, A., Russell, L.D., Grothe, C., Rudolf, M. and Gratzl, M. (1991) Endocrinology 129, 921–924). We have now addressed the question whether this 30 kDa bFGF-like protein is a large bFGF form and whether it is produced by pachytene spermatocytes. We detected bFGF mRNA in homogenates of isolated mouse spermatocytes (which consisted mainly of pachytene spermatocytes) using S1 nuclease protection assays. As shown by Western blot analyses, the bFGF mRNA in mouse spermatocytes is translated into bFGF of an approximate molecular weight of 30 kDa. Neither bFGF mRNA, nor bFGF itself, was observed in isolated mouse Leydig cells. These results indicate that the immunoreactive bFGF-like protein observed previously in germ cells of the murine testis is identical to bFGF. Thus, germ cells of the testis produce bFGF, which may exert regulatory function in the process of spermatogenesis

Myelination in the absence of UDP-galactose:ceramide galactosyl-transferase and fatty acid 2 -hydroxylase

<p>Abstract</p> <p>Background</p> <p>The sphingolipids galactosylceramide (GalCer) and sulfatide are major myelin components and are thought to play important roles in myelin function. The importance of GalCer and sulfatide has been validated using UDP-galactose:ceramide galactosyltransferase-deficient (<it>Cgt</it>^-/-) mice, which are impaired in myelin maintenance. These mice, however, are still able to form compact myelin. Loss of GalCer and sulfatide in these mice is accompanied by up-regulation of 2-hydroxylated fatty acid containing (HFA)-glucosylceramide in myelin. This was interpreted as a partial compensation of the loss of HFA-GalCer, which may prevent a more severe myelin phenotype. In order to test this hypothesis, we have generated <it>Cgt</it>^-/-mice with an additional deletion of the fatty acid 2-hydroxylase (<it>Fa2h</it>) gene.</p> <p>Results</p> <p><it>Fa2h</it>^-/-/Cgt^-/-double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to <it>Cgt</it>^-/-mice the amount of GlcCer in CNS myelin was strongly reduced in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice did not differ significantly from that of <it>Cgt</it>^-/-mice, and there was no obvious phenotypic difference between <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-and <it>Cgt</it>^-/-mice</p> <p>Conclusions</p> <p>These data show that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid and suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in <it>Cgt</it>^-/-mice does not functionally compensate the loss of HFA-GalCer.</p

Peripheral nerve regeneration through hydrogel enriched chitosan conduits containing engineered Schwann cells for drug delivery

Critical length nerve defects in the rat sciatic nerve model were reconstructed with chitosan nerve guides filled with Schwann cells (SCs) containing hydrogel. The transplanted SCs were naive or had been genetically modified to overexpress neurotrophic factors, thus providing a cellular neurotrophic factor delivery system. Prior to the assessment in vivo, in vitro studies evaluating the properties of engineered SCs overexpressing glial cell line-derived neurotrophic factor (GDNF) or fibroblast growth factor 2 (FGF-218kDa) demonstrated their neurite outgrowth inductive bioactivity for sympathetic PC-12 cells as well as for dissociated dorsal root ganglion cell drop cultures. SCs within NVR-hydrogel, which is mainly composed of hyaluronic acid and laminin, were delivered into the lumen of chitosan hollow conduits with a 5% degree of acetylation. The viability and neurotrophic factor production by engineered SCs within NVR-Gel inside the chitosan nerve guides was further demonstrated in vitro. In vivo we studied the outcome of peripheral nerve regeneration after reconstruction of 15-mm nerve gaps with either chitosan/NVR-Gel/SCs composite nerve guides or autologous nerve grafts (ANGs). While ANGs did guarantee for functional sensory and motor regeneration in 100% of the animals, delivery of NVR-Gel into the chitosan nerve guides obviously impaired sufficient axonal outgrowth. This obstacle was overcome to a remarkable extent when the NVR-Gel was enriched with FGF-218kDa overexpressing SCs

Correction to “In Vitro Assessment of Synthetic Nano Engineered Graft Designed for Further Clinical Study in Nerve Regeneration”

In the article entitled “In vitro assessment of synthetic nano engineered graft designed for further clinical study in nerve regeneration” which published in International Clinical Neuroscience Journal 2018;5(3)86-91, Figure 6 contained some errors, in which panels A, C, and D have been reported mistakenly. This mistake happened inadvertently during the data processing and data collection. The corrected Figure 6 and corresponding caption is presented in the present correction, in which the correct SEM images replaces in panels A, C, and D. The reader should note that this error does not affect the scientific quality of the work and no other section of the manuscript is affected by this error. In addition, the correction does not affect the conclusions of that paper. We would like to apologise for any inconvenience caused

In Vitro Assessment of Synthetic Nano Engineered Graft Designed for Further Clinical Study in Nerve Regeneration

Background: Electrospun nanofibrous scaffolds are considered as promising candidates in neural tissue regeneration due to their ability to support neural cell attachment, spreading and proliferation.Methods: In this paper, various type of nanofibers scaffold based on polycaprolactone) (PCL) were fabricated using electrospinning. The main drawback of PCL scaffolds is their low bioactivity of scaffold surface. To overcome this surface and composition modification was used to enhanced hydrophilicity and bioactivity of scaffold.Results: The scanning electron microscopy (SEM) results indicate that fiber diameter entirely depends on the solvent system and added component of gelatin and chitosan which by adding gelatin and chitosan fiber diameter decreased. In vitro studies using PC12 cells revealed that the plasma surface modified and blended scaffold with chitosan and gelatin nanofibrous scaffold supports cell attachment, spreading and indicate a significant increase in proliferation of PC12 in the presence of chitosan. The results demonstrated that gelatin and chitosan caused a significant enhancement in the bioactivity of the scaffold, which confirmed by MTT assay and improved the cell spreading and proliferation of neural cell on the scaffolds.Conclusion: Based on the experimental results, the PCL/chitosan/PPy conductive substrate could be used as a potential scaffold for clinical research in the field of neural regeneration and healing

In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)-immortalized, neonatal, and adult-as well as rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were analyzed with regard to their cell metabolic activity, proliferation profiles, and cell morphology after different time points of mono-and cocultures on the chitosan films. Overall the results demonstrate a good cytocompatibility of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell-biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts.This work was supported by the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement No. 278612. This work was also co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), ao abrigo do Quadro de Referencia Estrategico Nacional (QREN), and atraves do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors gratefully acknowledge the delivery of the chitosan raw material by Altakitin S.A., Portugal, and the fabrication of chitosan films by Medovent GmbH, Germany

Schwann Cells Overexpressing FGF-2 Alone or Combined with Manual Stimulation Do Not Promote Functional Recovery after Facial Nerve Injury

Purpose. To determine whether transplantation of Schwann cells (SCs) overexpressing different isoforms of fibroblast growth factor 2 (FGF-2) combined with manual stimulation (MS) of vibrissal muscles improves recovery after facial nerve transection in adult rat. Procedures. Transected facial nerves were entubulated with collagen alone or collagen plus naïve SCs or transfected SCs. Half of the rats received daily MS. Collateral branching was quantified from motoneuron counts after retrograde labeling from 3 facial nerve branches. Quality assessment of endplate reinnervation was combined with video-based vibrissal function analysis. Results. There was no difference in the extent of collateral axonal branching. The proportion of polyinnervated motor endplates for either naïve SCs or FGF-2 over-expressing SCs was identical. Postoperative MS also failed to improve recovery. Conclusions. Neither FGF-2 isoform changed the extent of collateral branching or polyinnervation of motor endplates; furthermore, this motoneuron response could not be overridden by MS

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