Decellularisation and Histological Characterisation of Porcine Peripheral Nerves

Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC’s) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC’s are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1 %; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1 % (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95 % (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair

In Vivo Gene Knockdown in Rat Dorsal Root Ganglia Mediated by Self-Complementary Adeno-Associated Virus Serotype 5 Following Intrathecal Delivery

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4–L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons

Acute- and late-phase matrix metalloproteinase (MMP)-9 activity is comparable in female and male rats after peripheral nerve injury

Abstract Background In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. Methods Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. Results The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. Conclusion The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes