Arash Moradzadeh

Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve.

Quantitative histomorphometry is the current gold standard for objective measurement of nerve architecture and its components. Many methods still in use rely heavily upon manual techniques that are prohibitively time consuming, predisposing to operator fatigue, sampling error, and overall limited reproducibility. More recently, investigators have attempted to combine the speed of automated morphometry with the accuracy of manual and semi-automated methods. Systematic refinements in binary imaging analysis techniques combined with an algorithmic approach allow for more exhaustive characterization of nerve parameters in the surgically relevant injury paradigms of regeneration following crush, transection, and nerve gap injuries. The binary imaging method introduced here uses multiple bitplanes to achieve reproducible, high throughput quantitative assessment of peripheral nerve. Number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris can be quantitatively evaluated with stratification of raw data by nerve component. Results of this semi-automated method are validated by comparing values against those obtained with manual techniques. The use of this approach results in more rapid, accurate, and complete assessment of myelinated axons than manual techniques.

The impact of motor and sensory nerve architecture on nerve regeneration.

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerves Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p<0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p<0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.

Ropivacaine-induced peripheral nerve injection injury in the rodent model.

BACKGROUND: Intraneural administration of local anesthetics has been associated with nerve damage. We undertook the present study to investigate histological changes induced by ropivacaine injection into rat sciatic nerve. METHODS: Fifty-four adult male Lewis rats were randomly distributed into 9 groups, 6 animals per group. Fifty microliters of normal saline, 10% phenol, or 0.75% ropivacaine were administered by intrafascicular injection, extrafascicular injection, or extraneural (topical) placement. At 2 weeks, animals were killed and the sciatic nerve at the injection site was evaluated with light microscopy, quantitative histomorphometry, and electron microscopy. RESULTS: On cross-sectional evaluation, extrafascicular ropivacaine injection and extraneural placement of ropivacaine were both associated with damage to the perineurium, with focal demyelination surrounded by edematous endoneurium. Intrafascicular injection of ropivacaine resulted in a wedge-shaped region of demyelination and focal axonal loss with some regeneration, bordered by a region of normally myelinated axons in a background of edematous endoneurium. Extrafascicular injection resulted in more significant damage than extraneural placement of ropivacaine, but less than intrafascicular injection as shown with quantitative histomorphometry. Quantitatively, ropivacaine-injured specimens had significantly lower nerve density than saline-injured specimens. Wallerian degeneration and perineural edema were also demonstrated qualitatively with electron microscopy. CONCLUSIONS: This study demonstrates that, in the rat model, ropivacaine is associated with marked histological abnormality, including edema of the perineurium and axonal destruction with wallerian degeneration, when injected into or extraneurally placed onto a nerve. Extrafascicular injection and extraneural placement were associated with similar, although milder, histological damage than intrafascicular injection. Further work is needed to investigate the functional implications, if any, of the histological abnormalities observed in this study.

Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration

Accurate assessment of peripheral nerve regeneration requires fixation techniques that preserve tissue in a natural state with minimal artifact. While transcardial perfusion fixation is accepted as the gold standard for tissue fixation, the less cumbersome approach of immersion fixation has been criticized for introducing artifacts in brain tissue. We investigated whether immersion fixation increased artifact compared to perfusion fixation in the rat sciatic nerve. Eighteen Lewis rats were randomized into three groups: glutaraldehyde immersion fixation; glutaraldehyde transcardial perfusion; and paraformaldehyde transcardial perfusion. All animals underwent sciatic nerve transection and repair followed by tissue harvest and fixation at three weeks. Qualitative assessment of neural architecture and histological features was followed by quantitative analysis of nerve regeneration parameters. Outcome measures included quantitative histomorphometry, analysis of axon/myelin ratios, assessment of fiber distributions, and ultrastructural analysis. No qualitative or quantitative differences were observed with immersion fixation when compared to the transcardial perfusion fixation methods. Immersion fixation is a valid method for assessment of peripheral nerve regeneration in a rat model.

Bipolar Electrocautery: A Rodent Model of Sunderland Third-degree Nerve Injury

OBJECTIVE To determine the Sunderland classification of a bipolar electrocautery injury. METHODS Twenty-two rats received crush (a reproducible Sunderland second-degree injury) or bipolar electrocautery injury and were evaluated for functional, histomorphometric, and immunohistochemical recovery at 21 or 42 days. Animal experiments were performed between July 3 and December 12, 2007. Axonal regeneration and end plate reinnervation were evaluated in double transgenic cyan fluorescent protein-conjugated Thy1 and green fluorescent protein-conjugated S100 mice. RESULTS Compared with crush injury, bipolar electrocautery injury caused greater disruption of myelin and neurofilament architecture at the injury site and decreased nerve fiber counts and percentage of neural tissue distal to the injury (P =.007). Complete functional recovery was seen after crush but not bipolar electrocautery injury. Serial live imaging demonstrated axonal regeneration at week 1 after crush and at week 3 after bipolar electrocautery injury. Qualitative assessment of motor end plate reinnervation at 42 days demonstrated complete neuromuscular end plate reinnervation in the crush group and only limited reinnervation in the bipolar electrocautery group. CONCLUSION Bipolar electrocautery injury in a rodent model resulted in a Sunderland third-degree injury, characterized by gradual, incomplete recovery without intervention.

Treatment Modality Affects Allograft-Derived Schwann Cell Phenotype and Myelinating Capacity

We used peripheral nerve allografts, already employed clinically to reconstruct devastating peripheral nerve injuries, to study Schwann cell (SC) plasticity in adult mice. By modulating the allograft treatment modality we were able to study migratory, denervated, rejecting, and reinnervated phenotypes in transgenic mice whose SCs expressed GFP under regulatory elements of either the S100b (S100-GFP) or nestin (Nestin-GFP) promoters. Well-differentiated SCs strongly expressed S100-GFP, while Nestin-GFP expression was stimulated by denervation, and in some cases, axons were constitutively labeled with CFP to enable in vivo imaging. Serial imaging of these mice demonstrated that untreated allografts were rejected within 20 days. Cold preserved (CP) allografts required an initial phase of SC migration that preceded axonal regeneration thus delaying myelination and maturation of the SC phenotype. Mice immunosuppressed with FK506 demonstrated mild subacute rejection, but the most robust regeneration of myelinated and unmyelinated axons and motor endplate reinnervation. While characterized by fewer regenerating axons, mice treated with the co-stimulatory blockade (CSB) agents anti-CD40L mAb and CTLAIg-4 demonstrated virtually no graft rejection during the 28 day experiment, and had significant increases in myelination, connexin-32 expression, and Akt phosphorylation compared with any other group. These results indicate that even with SC rejection, nerve regeneration can occur to some degree, particularly with FK506 treatment. However, we found that co-stimulatory blockade facilitate optimal myelin formation and maturation of SCs as indicated by protein expression of myelin basic protein (MBP), connexin-32 and phospho-Akt.

Induction of regional collateral sprouting following muscle denervation.

Objectives/Hypothesis: Anecdotal clinical findings suggest that denervated muscle may regain modest functional recovery via spontaneous collateral sprouts from intact adjacent nerve fibers. The current study evaluates the conditions needed for the denervated masseter muscle to induce axonal sprouting from the facial nerve. We hypothesize that epineurial injury is required to induce collateral sprouting toward a neighboring denervated muscle.

R416 – Bipolar Cautery: Sunderland Third Degree Nerve Injury Model

Problem The prognosis of bipolar electrocautery nerve injury is unpredictable because few experiments compare it to characterized nerve injury models. Methods 16 Lewis rats were randomized to sciatic crush or bipolar cautery and at 21 days evaluated with nerve morphometry and walking tack analysis. Double transgenic Thy1-CFP/S100-GFP mice were used to serially image axonal regeneration and Schwann cells (SC) over time following injury. Results In rats, bipolar cautery injury shows greater disruption of myelin and neurofilament architecture at the injury site. There is decreased total nerve fiber counts distal to the injury (p<0.05). Walking track analysis demonstrates functional recovery after crush, but not after cautery injury. Serial imaging of mice, shows axonal regeneration starting at week 1 after crush, but late, partial axonal regeneration in the cautery group; these findings were reflected in endplate reinnervation at 42 days. Conclusion Results suggest that a Sunderland type 3 injury, characterized by slow, variable, incomplete recovery, results from a bipolar electrocautery injury. Significance Bipolar cautery injuries can be observed without immediate surgical intervention.

P002: Motor Nerve Architecture and Peripheral Nerve Regeneration

OBJECTIVES: 1. Learn how to apply an image-guided technique to repair frontal sinus fractures. 2. Be able to simplify locating of the frontal sinus for open repair of fractures. METHODS: A 25-year-old male presented with a comminuted anterior table frontal sinus fracture with minimally displaced posterior table. There was no evidence of cerebrospinal fluid leak or intracranial injury. Patient desired open reduction and internal fixation of the depressed anterior table. After raising a bicoronal flap, image guidance was used to confirm the location of the fracture. RESULTS: Using a novel postioning of the image guidance headset the team was able to successfully locate the position of the frontal sinus fracture without inhibiting access for the bicoronal incision. The fracture was successfully reduced and plated without postoperative complications. CONCLUSIONS: Using image guidance technology helps identify the location of the frontal sinus fracture and helps identify the extent of the osteoplastic flap. Using a novel positioning system of the image guidance headset enabled the surgeons to have full access for a bicoronal incision and direct access to the forehead. This technique overcomes the shortcomings of the imprecise plain film Caldwell views or trans sinus illumination techniques.