Improving interfaces for nerve repair

Autologous nerve grafts are the current gold standard for the repair of peripheral nerve injuries. However, there is a need to develop an alternative to this technique, as donor-site morbidities such as neuroma formation and permanent loss of function are a few of the limitations concerned with this technique. Artificial nerve conduits have therefore emerged as an alternative for the repair of short peripheral nerve defects of less than 30 mm, however they do not surpass autologous nerve grafts clinically. To develop a nerve conduit that supports regeneration over long nerve gaps and in large diameter nerves, researchers have focused on functionalizing of the conduits by studying the components that enhance nerve regeneration such as micro/nano-topography, growth factor delivery systems, supportive cells and extracellular matrix (ECM) proteins as well as understanding the complex biological reactions that take place during peripheral nerve regeneration. This thesis presents strategies to improve peripheral nerve interfaces to better the regenerative potential by using dorsal root ganglions (DRGs) isolated from neonatal rats as an in vitro model of nerve regeneration. The work started off by investigating the usefulness of a frog foam protein Ranaspumin-2 (Rsn2) to coat biomaterials for compatibility, this lead to the discovery of temporary cell adhesion on polydimethylsiloxane (PDMS), which was investigated as a suitable tool to derive cell-sheets for nerve repair. The influence of Rsn2 anchored to specific adhesion peptide sequences, such as isoleucine-lysine-valine-alanine-valine (IKVAV), a sequence derived from laminin proven to promote cell adhesion and neurite outgrowth, was tested as a useful means to influence nerve regeneration. This approach improves the axonal outgrowth and maintains outgrowth long term. Based on the hypothesis that combinational modulation of substrate topography, stiffness and neurotrophic support, affects axonal outgrowth in whole DRGs, dissociated DRGs were used to assess if these factors similarly act at the single cell level. Rho associated protein kinase (ROCK) and myosin II inhibitors, which affect cytoskeletal contractility, were used to influence growth cone traction forces and have shown that these factors work in combination by interfering with growth cone dynamic creating a different response in axonal outgrowth at the single cell level.

Friedreich Ataxia

Friedreich's ataxia (FRDA) is the most common autosomal recessive hereditary ataxia in Caucasians. Neurological symptoms dominate the clinical picture. The underlying neuropathology affects the dorsal root ganglia, the spinal cord, and the deep cerebellar nuclei. In addition, most cases present a hypertrophic cardiomyopathy that may cause premature death. Other problems include a high risk of diabetes, skeletal abnormalities such as kyphoscoliosis, and pes cavus. Most patients carry a homozygous expansion of GAA trinucleotide repeat within the first intron of the FXN gene, leading to repressed transcription through epigenetic mechanisms. The encoded protein, frataxin, is localized in mitochondria and participates in the biogenesis of iron-sulfur clusters. Frataxin deficiency leads to mitochondrial dysfunction, altered iron metabolism, and oxidative damage. Thanks to progress in understanding pathogenesis and to the development of animal and cellular models, therapies targeted to correct frataxin deficiency or its downstream consequences are being developed and tested in clinical trials.

Postnatal development of the retina in root vole Microtus oeconomus

Vision plays an important role in the living habits of animals, especially in feeding. We investigated the postnatal development of retina in root vole Microtus oeconornus. The result shows that the retina of the M. oeconornus is very primitive before postnatal day （PD） 3. The neuroblastic layer does not differentiate and makes up more than half of the retina layer. The outer plexiform layer （OPL） first comes into existence at PDS. At PD6, as the presence of the OPL becomes obvious, the outer nuclear layer （ONL） and inner nuclear layer （INL） are much clearer. At PD18, the retina is similar to an adult retina and each layer becomes distinct. The thickness and cell density of the ganglion cell layer （GCL） and ONL during different postnatal days were also examined. These results show that the thickness and density of ONL increase during ontogeny, while the thickness and density of GCL decrease. Compared with Rattus norvegicus, Apodemus agrarius , Cricetulus triton, Microtus mandarinus , Myospalax cansus , Spermophilus dauricus and Sciurotamias davidianus, the histological structure of the retina of M. oeconornus is between that of nocturnal and diurnal rodents.

Evidence of reciprocal connections between the dorsal raphe nucleus and the retina in the monkey Cebus apella

Possible connections between the retina and the raphe nuclei were investigated in the monkey Cebus apella by intraocular injection of cholera toxin B subunit (CTb). CTb-positive fibers were seen in the lateral region of the dorsal raphe nucleus (DR) on the side contralateral to the injection, and a few labeled perikarya were observed in the lateral portion of the DR on the ipsilateral side. Our findings suggest that direct and reciprocal connections between the retina and DR may exist in Cebus apella. These connections might be part of an important pathway through which the light/dark cycle influences the Activity and/or functional status of raphe neurons, with potential effects on a broad set of neural and behavioral circuits. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Comparison of the biocompatibility of different root canal irrigants

The purpose of this study was to compare the reaction of rat subcutaneous connective tissue to 0.9% sterile saline, 2.5% sodium hypochlorite (NaOCl), 5.25% NaOCl and 2% chlorhexidine gluconate solution or gel. Six circles were demarcated on the dorsal skin of 24 male Wistar rats, leaving 2 cm between each circle. Using a syringe, 0.1 mL of each root canal irrigant was injected subcutaneously into 5 circles. In the 6th circle, the needle of an empty syringe was introduced into the skin, but no irrigant was injected (control group). Evaluations were undertaken at 2 h, 48 h, 14 days and 30 days post-procedure. Tissue samples were excised, embedded in paraffin blocks and 3-mu m-thick sections were obtained and stained with hematoxylin and eosin. The areas of inflammatory reaction were evaluated and analyzed statistically by ANOVA and Tukey's test. The control group showed few or no inflammatory reaction areas in the subcutaneous tissue. 0.9% saline solution, 2.0% chlorhexidine solution and 2.5% NaOCl showed a good biocompatibility, as very mild inflammatory reaction was detected at 14 days and tissue repair occurred at 30 days. 5.25% NaOCl was the most toxic irrigant, as the number of inflammatory cells remained elevated at 14 and 30 days. The group treated with 2.0% chlorhexidine gluconate gel presented a moderate inflammatory response at 14 days, which decreased at 30 days, being considered similar to that of the control group, 0.9% saline solution, 2.0% chlorhexidine solution and 2.5% NaOCl at this experimental period.

Evaluation of the biocompatibility of root canal sealers using subcutaneous implants

The purpose of this study was to evaluate in vivo the biocompatibility of Endométhasone, Pulp Canal Sealer EWT and AHPlus root canal sealers after implantation in rat connective tissue. Twenty-four Wistar-Furth rats were used. Polyethylene tubes were filled with the sealers and implanted into specific dorsal subdermal tissue sites of the rats. Implants were removed after 3, 7 and 30 days, fixed and processed for glycol methacrylate-embedding technique to be examined microscopically. On the 3rd day, there was a mild inflammatory reaction to Pulp Canal Sealer EWT implants, but a severe response to the other sealers with presence of acute inflammatory cells. On the 7th day, tissue organization was more evident with attenuation of the inflammatory reaction, especially for the AH-Plus implants. On the 30th day, connective tissue with few inflammatory cells was observed in contact with all sealer implants. In this time interval, the tissue in contact with Pulp Canal Sealer EWT implants was more organized, while the tissue close to Endométhasone and AH-Plus implants showed a mild persistent inflammatory reaction and had similar results to each other. In conclusion, the sealers had a similar pattern of irritation, which was more severe in the beginning and milder with time, in such a way that all sealers showed a persistent mild reaction. Pulp Canal Sealer EWT yielded better tissue organization than Endométhasone and AH-Plus, which, in turn, showed similar results to each other.

Biocompatibility and biomineralization assessment of a new root canal sealer and root-end filling material

Objective: This study investigated the short-term subcutaneous tissue reaction and biomineralization ability of two epoxy-based root canal sealers containing calcium hydroxide (MBP and MBPc) and ProRoot MTA. Materials and methods: Polyethylene tubes containing the materials were implanted into the dorsal connective tissue of Wistar rats (n = 52) for 7 or 30 days; empty implanted tubes served as controls. Specimens were stained with hematoxylin-eosin and von Kossa stain or left unstained for observation under polarized light. Qualitative and quantitative evaluations of all tissue reactions were performed. One-way anova and the Kruskal-Wallis test were used for statistical analysis (P < 0.05). Results: No significant differences were observed among the groups. All three materials induced mild-to-moderate tissue reactions at 7 days, which decreased over time. Dystrophic mineralization and birefringent structures were observed only in the ProRoot MTA ® group. Conclusion: Both MBP and MBPc appear to be biocompatible but do not stimulate biomineralization. © 2012 John Wiley & Sons A/S.

MRI for differentiating ganglion and synovitis in the chronic painful wrist

OBJECTIVE: The purpose of our study was to determine if preoperative MRI can differentiate between occult ganglion and synovitis in the chronic painful wrist. CONCLUSION: MRI is accurate in preoperatively distinguishing between ganglion and synovitis in the setting of chronic dorsal wrist pain. Four main criteria were useful: margin, shape, internal structure, and enhancement after administration of contrast material, with shape and internal structure being most helpful.

Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

Increased intraoperative epidural pressure in lumbar spinal stenosis patients with a positive nerve root sedimentation sign

Purpose The sedimentation sign (SedSign) has been shown to discriminate well between selected patients with and without lumbar spinal stenosis (LSS). The purpose of this study was to compare the pressure values associated with LSS versus non-LSS and discuss whether a positive SedSign may be related to increased epidural pressure at the level of the stenosis. Methods We measured the intraoperative epidural pressure in five patients without LSS and a negative SedSign, and in five patients with LSS and a positive SedSign using a Codman TM catheter in prone position under radioscopy. Results Patients with a negative SedSign had a median epidural pressure of 9 mmHg independent of the measurement location. Breath and pulse-synchronous waves accounted for 1–3 mmHg. In patients with monosegmental LSS and a positive SedSign, the epidural pressure above and below the stenosis was similar (median 8–9 mmHg). At the level of the stenosis the median epidural pressure was 22 mmHg. A breath and pulse-synchronous wave was present cranial to the stenosis, but absent below. These findings were independent of the cross-sectional area of the spinal canal at the level of the stenosis. Conclusions Patients with LSS have an increased epidural pressure at the level of the stenosis and altered pressure wave characteristics below. We argue that the absence of sedimentation of lumbar nerve roots to the dorsal part of the dural sac in supine position may be due to tethering of affected nerve roots at the level of the stenosis.

Sleep modifies retinal ganglion cell responses in the normal rat

Recordings were obtained from the visual system of rats as they cycled normally between waking (W), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Responses to flashes delivered by a light-emitting diode attached permanently to the skull were recorded through electrodes implanted on the cornea, in the chiasm, and on the cortex. The chiasm response reveals the temporal order in which the activated ganglion cell population exits the eyeball; as reported, this triphasic event is invariably short in latency (5–10 ms) and around 300 ms in duration, called the histogram. Here we describe the differences in the histograms recorded during W, SWS, and REM. SWS histograms are always larger than W histograms, and an REM histogram can resemble either. In other words, the optic nerve response to a given stimulus is labile; its configuration depends on whether the rat is asleep or awake. We link this physiological information with the anatomical fact that the brain dorsal raphe region, which is known to have a sleep regulatory role, sends fibers to the rat retina and receives fibers from it. At the cortical electrode, the visual cortical response amplitudes also vary, being largest during SWS. This well known phenomenon often is explained by changes taking place at the thalamic level. However, in the rat, the labile cortical response covaries with the labile optic nerve response, which suggests the cortical response enhancement during SWS is determined more by what happens in the retina than by what happens in the thalamus.

Rapid Atrophy of the Lumbar Multifidus Follows Experimental Disc or Nerve Root Injury.

Study Design. Experimental study of muscle changes after lumbar spinal injury. Objectives. To investigate effects of intervertebral disc and nerve root lesions on cross-sectional area, histology and chemistry of porcine lumbar multifidus. Summary of Background Data. The multifidus cross-sectional area is reduced in acute and chronic low back pain. Although chronic changes are widespread, acute changes at 1 segment are identified within days of injury. It is uncertain whether changes precede or follow injury, or what is the mechanism. Methods. The multifidus cross-sectional area was measured in 21 pigs from L1 to S1 with ultrasound before and 3 or 6 days after lesions: incision into L3 - L4 disc, medial branch transection of the L3 dorsal ramus, and a sham procedure. Samples from L3 to L5 were studied histologically and chemically. Results. The multifidus cross-sectional area was reduced at L4 ipsilateral to disc lesion but at L4 - L6 after nerve lesion. There was no change after sham or on the opposite side. Water and lactate were reduced bilaterally after disc lesion and ipsilateral to nerve lesion. Histology revealed enlargement of adipocytes and clustering of myofibers at multiple levels after disc and nerve lesions. Conclusions. These data resolve the controversy that the multifidus cross-sectional area reduces rapidly after lumbar injury. Changes after disc lesion affect 1 level with a different distribution to denervation. Such changes may be due to disuse following reflex inhibitory mechanisms.

Combining ganglion cell topology and data of patients with glaucoma to determine a structure-function map

PURPOSE: To introduce techniques for deriving a map that relates visual field locations to optic nerve head (ONH) sectors and to use the techniques to derive a map relating Medmont perimetric data to data from the Heidelberg Retinal Tomograph. METHODS: Spearman correlation coefficients were calculated relating each visual field location (Medmont M700) to rim area and volume measures for 10 degrees ONH sectors (HRT III software) for 57 participants: 34 with glaucoma, 18 with suspected glaucoma, and 5 with ocular hypertension. Correlations were constrained to be anatomically plausible with a computational model of the axon growth of retinal ganglion cells (Algorithm GROW). GROW generated a map relating field locations to sectors of the ONH. The sector with the maximum statistically significant (P < 0.05) correlation coefficient within 40 degrees of the angle predicted by GROW for each location was computed. Before correlation, both functional and structural data were normalized by either normative data or the fellow eye in each participant. RESULTS: The model of axon growth produced a 24-2 map that is qualitatively similar to existing maps derived from empiric data. When GROW was used in conjunction with normative data, 31% of field locations exhibited a statistically significant relationship. This significance increased to 67% (z-test, z = 4.84; P < 0.001) when both field and rim area data were normalized with the fellow eye. CONCLUSIONS: A computational model of axon growth and normalizing data by the fellow eye can assist in constructing an anatomically plausible map connecting visual field data and sectoral ONH data.