isolation-culture-characterization-primary-schwann-cells

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

The Pennsylvania State University College of Medicine

The 6-mercaptopurine (6-MP) is an effective immunosuppressant and anti-cancer drug. However, the usage of 6-MP is limited due to its well-known side effects, such as myelotoxicity and hepato-renal toxicity. To curtail the potential toxic effects, we have used chitosan as a natural biodegradable and biocompatible polysaccharide to synthesize 6-Mercaptopurine-Chitosan Nanoparticles (6-MP-CNPs).

Toxicity evaluation of 6-mercaptopurine-Chitosan nanoparticles in rats.

The functional recovery following non-severing peripheral nerve injury (PNI) is often incomplete. Erythropoietin (EPO) is a pleiotropic hormone and it has been shown to protect peripheral nerves following mild and even moderate severity injuries. However, the effectiveness of EPO in severe PNI is largely unknown. In this study, we sought to investigate the neuroprotective effect of a new dose regimen of EPO in severe sciatic nerve crush injury (SSCI). Adult male mice (8 animals/group) were randomly assigned to sham (normal saline, 0.1 ml/mouse), SSCI (normal saline, 0.1 ml/mouse) and SSCI with EPO (5000 IU/kg) groups. SSCI was performed using calibrated forceps for 30 sec. EPO or normal saline was administered intraperitoneally immediately after the SSCI and at post-injury day1 and 2. The functional recovery after injury was assessed by sciatic function index (SFI), von Frey Test (VFT), and grip strength test. Mice were euthanized on day 7 and 21 and nerves at injury/peri-injury site were processed for gene (quantitative real-time PCR) and protein (immunohistochemistry) expression analysis. EPO significantly improved SFI, VFT, and hind limb paw grip strength from post-injury day 7. EPO demonstrated significant regulatory effects on mRNA expression of inflammatory (IL-1β and TNF-α), anti-inflammatory (IL-10), angiogenesis (VEGF and eNOS), and myelination (MBP) genes. The protein expression of IL-1β, F4/80, CD31, NF-κB p65, NF-H, MPZ, and DHE (redox-sensitive probe) was also significantly modulated by EPO treatment. In conclusion, the new dose regimen of EPO augments sciatic nerve functional recovery by mitigating inflammatory, anti-inflammatory, oxidative stress, angiogenesis, and myelination components of SSCI.

An effective erythropoietin dose regimen protects against severe nerve injury-induced pathophysiological changes with improved neural gene expression and enhances functional recovery.

Erythropoietin (EPO) promotes myelination and functional recovery in rodent peripheral nerve injury (PNI). While EPO receptors (EpoR) are present in Schwann cells, the role of EpoR in PNI recovery is unknown because of the lack of EpoR antagonists or Schwann cell-specific EpoR knockout animals.

Obligatory role of Schwann cell-specific erythropoietin receptors in erythropoietin-induced functional recovery and neurogenic muscle atrophy after nerve injury.

Peripheral nerve transection is associated with permanent functional deficit even after advanced microsurgical repair. While it is difficult to investigate the reasons of poor functional outcomes of microsurgical repairs in humans, we developed a novel pre-clinical nerve transection method that allows reliable evaluation of nerve regeneration, neural angiogenesis, muscle atrophy, and functional recovery. Adult male C57BL/6 mice were randomly assigned to four different types of sciatic nerve transection: Simple Transection (ST), Simple Transection & Glue (TG), Stepwise Transection and Sutures (SU), and Stepwise Transection and Glue (STG). Mice were followed for 28 days for sciatic function index (SFI), and sciatic nerves and hind limb muscles were harvested for histomorphological and cellular analyses. Immunohistochemistry revealed more directional nerve fiber growth in SU and STG groups compared with ST and TG groups. Compared to ST and TG groups, optimal neural vessel density and branching index in SU and STG groups were associated with significantly decreased muscle atrophy, increased myofiber diameter, and improved SFI. In conclusion, our novel STG method represents an easily reproducible and reliable model with close resemblance to the pathophysiological characteristics of SU model, and this can be easily reproduced by any lab.

A novel nerve transection and repair method in mice: histomorphometric analysis of nerves, blood vessels, and muscles with functional recovery.

Peripheral nerve crush injury (PNCI) models are commonly used to study nerve damage and the potential beneficial effects of novel therapeutic strategies. Current models of PNCI rely on inter-device and operator precision to limit the variation with applied pressure. Although the inability to accurately quantify the PNCI pressure may result in reduced reproducibility between animals and studies, there is very limited information on the standardization and quantification of applied pressure with PNCI. To address this deficit, we constructed a novel device comprised of an Arduino UNO microcontroller board and Force Sensitive Resistor capable of reporting the real-time pressure applied to a nerve.

Novel Real-time Digital Pressure Sensor Reveals Wide Variations in Current Nerve Crush Injury Models.

Functional recovery following primary nerve repair of a transected nerve is often poor even with advanced microsurgical techniques. Recently, we developed a novel sciatic nerve transection method where end-to-end apposition of the nerve endings with minimal gap was performed with fibrin glue. We demonstrated that transected nerve repair with gluing results in optimal functional recovery with improved axonal neurofilament distribution profile compared to the end-to-end micro-suture repair. However, the impact of axonal misdirection and misalignment of nerve fascicles remains largely unknown in nerve-injury recovery. We addressed this issue using a novel nerve repair model with gluing.

Purposeful Misalignment of Severed Nerve Stumps in a Standardized Transection Model Reveals Persistent Functional Deficit With Aberrant Neurofilament Distribution.

Traumatic peripheral nerve injury (TPNI) represents a major medical problem that results in loss of motor and sensory function, and in severe cases, limb paralysis and amputation. To date, there are no effective treatments beyond surgery in selective cases. In repurposing studies, we found that daily systemic administration of the FDA-approved drug 4-aminopyridine (4-AP) enhanced functional recovery after acute peripheral nerve injury. This study was aimed at constructing a novel local delivery system of 4-AP using thermogelling polymers. We optimized a thermosensitive (4-AP)-poly(lactide--glycolide)--poly(ethylene glycol)--poly(lactide--glycolide) (PLGA-PEG-PLGA) block copolymer formulation. (4-AP)-PLGA-PEG exhibited controlled release of 4-AP both  and  for approximately 3 weeks, with clinically relevant safe serum levels in animals. Rheological investigation showed that (4-AP)-PLGA-PEG underwent a solution to gel transition at 32 °C, a physiologically relevant temperature, allowing us to administer it to an injured limb while subsequently forming an  gel. A single local administration of (4-AP)-PLGA-PEG remarkably enhanced motor and sensory functional recovery on post-sciatic nerve crush injury days 1, 3, 7, 14, and 21. Moreover, immunohistochemical studies of injured nerves treated with (4-AP)-PLGA-PEG demonstrated an increased expression of neurofilament heavy chain (NF-H) and myelin protein zero (MPZ) proteins, two major markers of nerve regeneration. These findings demonstrate that (4-AP)-PLGA-PEG may be a promising long-acting local therapeutic agent in TPNI, for which no pharmacologic treatment exists.

(4-Aminopyridine)-PLGA-PEG as a Novel Thermosensitive and Locally Injectable Treatment for Acute Peripheral Nerve Injury.

Antibiotics (ABX) are widely used for life-threatening infections and also for routine surgical operations. Compelling evidence suggests that ABX-induced alterations of gut microbiota composition, termed dysbiosis, are linked with diverse disease states including neurological and neurodegenerative conditions. To combat the consequences of dysbiosis, probiotics (PBX) are widely used. ABX-induced dysbiosis is reported to impair neurological function after spinal cord injury. Traumatic peripheral nerve injury (TPNI) results in profound neurologic impairment and permanent disability. It is unknown whether ABX treatment-induced dysbiosis has any impact on TPNI-induced functional recovery, and if so, what role medical-grade PBX could have on TPNI recovery.

Altered gut microbiota composition with antibiotic treatment impairs functional recovery after traumatic peripheral nerve crush injury in mice: effects of probiotics with butyrate producing bacteria.

Following acute sciatic nerve crush injury (SNCI), inflammation and the improper phagocytic clearance of dying Schwann cells (SCs) has effects on remodeling that lead to morbidity and incomplete functional recovery. Therapeutic strategies like the use of erythropoietin (EPO) for peripheral nerve trauma may serve to bring immune cell phagocytotic clearance under control to support debris clearance. We evaluated EPO's effect on SNCI and found EPO treatment increased myelination and sciatic functional index (SFI) and bolstered anti-apoptosis and phagocytosis of myelin debris via CD206 macrophages when compared to saline treatment. EPO enhanced M2 phenotype activity, both in bone marrow-derived macrophages (BMMØs) and peritoneal-derived macrophages (PMØs) in vitro, as well as in PMØs in vivo. EPO increased efferocytosis of apoptotic sciatic nerve derived Schwann cells (SNSCs) in both settings as demonstrated using immunofluorescence (IF) and flow cytometry. EPO treatment significantly attenuated pro-inflammatory genes (IL1β, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) and the cell-surface marker CD206. EPO also increased anti-apoptotic (Annexin V/7AAD) effects after lipopolysaccharide (LPS) induction in macrophages. Our data demonstrate EPO promotes the M2 phenotype macrophages to ameliorate apoptosis and efferocytosis of dying SCs and myelin debris and improves SN functional recovery following SNCI.

Article	Year
Toxicity evaluation of 6-mercaptopurine-Chitosan nanoparticles in rats. Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society\| PubMed ID: 31933529	2020
An effective erythropoietin dose regimen protects against severe nerve injury-induced pathophysiological changes with improved neural gene expression and enhances functional recovery. International immunopharmacology\| PubMed ID: 32143001	2020
Obligatory role of Schwann cell-specific erythropoietin receptors in erythropoietin-induced functional recovery and neurogenic muscle atrophy after nerve injury. Muscle & nerve\| PubMed ID: 33205838	2021
A novel nerve transection and repair method in mice: histomorphometric analysis of nerves, blood vessels, and muscles with functional recovery. Scientific reports\| PubMed ID: 33303798	2020
Novel Real-time Digital Pressure Sensor Reveals Wide Variations in Current Nerve Crush Injury Models. Military medicine\| PubMed ID: 33499447	2021
Purposeful Misalignment of Severed Nerve Stumps in a Standardized Transection Model Reveals Persistent Functional Deficit With Aberrant Neurofilament Distribution. Military medicine\| PubMed ID: 33499508	2021
(4-Aminopyridine)-PLGA-PEG as a Novel Thermosensitive and Locally Injectable Treatment for Acute Peripheral Nerve Injury. ACS applied bio materials\| PubMed ID: 34142019	2021
Altered gut microbiota composition with antibiotic treatment impairs functional recovery after traumatic peripheral nerve crush injury in mice: effects of probiotics with butyrate producing ... BMC research notes\| PubMed ID: 35197129	2022
Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury. Cell death & disease\| PubMed ID: 35296651	2022

Prem Kumar Govindappa

Biography

JoVE Journal Publications

Other Publications