A simple and robust method for automating analysis of naïve and regenerating peripheral nerves

ABSTRACT: Background Manual axon histomorphometry (AH) is time- and resource-intensive, which has inspired many attempts at automation. However, there has been little investigation on implementation of automated programs for widespread use. Ideally such a program should be able to perform AH across imaging modalities and nerve states. AxonDeepSeg (ADS) is an open source deep learning program that has previously been validated in electron microscopy. We evaluated the robustness of ADS for peripheral nerve axonal histomorphometry in light micrographs prepared using two different methods. Methods Axon histomorphometry using ADS and manual analysis (gold-standard) was performed on light micrographs of naïve or regenerating rat median nerve cross-sections prepared with either toluidine-resin or osmium-paraffin embedding protocols. The parameters of interest included axon count, axon diameter, myelin thickness, and g-ratio. Results Manual and automatic ADS axon counts demonstrated good agreement in naïve nerves and moderate agreement on regenerating nerves. There were small but consistent differences in measured axon diameter, myelin thickness and g-ratio; however, absolute differences were small. Both methods appropriately identified differences between naïve and regenerating nerves. ADS was faster than manual axon analysis. Conclusions Without any algorithm retraining, ADS was able to appropriately identify critical differences between naïve and regenerating nerves and work with different sample preparation methods of peripheral nerve light micrographs. While there were differences between absolute values between manual and ADS, ADS performed consistently and required much less time. ADS is an accessible and robust tool for AH that can provide consistent analysis across protocols and nerve states

QS9: Determining the Critical Time of Chronic Schwann Cell Denervation on Functional Recovery and Rna Expression

PURPOSE: There is poor functional recovery following delayed peripheral nerve repair since both muscle and Schwann cells (SC) undergo denervation atrophy. We investigated the specific temporal effect of nerve/SC denervation on recovery as well as changes in RNA expression in the nerves that may elucidate changes in recovery potential. We hypothesized that functional recovery would be worse after prolonged nerve/SC denervation and that the expression profiles would differ. METHODS: Our study was conducted using a forelimb model in adult Lewis rats. Each animal underwent unilateral forelimb denervation of 8, 12, 16, or 24 weeks duration. In the functional recovery arm of the study, the ulnar nerve was denervated proximally or a sham surgery was performed. After the denervation period had elapsed, an in situ nerve transfer of median to ulnar to median nerve was performed. Functional recovery was then measured by stimulated grip strength weekly for 12 weeks. In the RNA expression arm, median and ulnar nerves were denervated. The same time points were used with the addition of a 1-week denervation group. After the denervation period, the median and ulnar nerves were harvested bilaterally. To create a comprehensive RNA-Seq dataset, the median nerve, with an average length of 3 cm, was homogenized and RNA was purified. RNA-sequencing was carried out using TrueSeq RiboZero gold kit. Samples were analyzed through FastQC, aligned to reference genome using STAR and quantified as transcripts per million (TPM) using Salmon. Principle component analysis was performed, followed by differential gene analysis using a linear mixed effects model to control for the control nerves being from the same animals. RESULTS: Functional recovery was statistically significantly different depending on the duration of nerve/SC denervation (P0.05, we identified 1624 genes differentially expressed, of which 327 genes were upregulated and rest (1297 genes) downregulated with denervation. CONCLUSIONS: Prolonged nerve/SC denervation of more than 12 weeks resulted in significantly worse functional recovery. RNA sequencing demonstrated that not only were there many genes differentially expressed, but these appear to vary with duration of denervation as well. Further investigation into the specific genes and their changes over time will allow us to know why recovery potential is decreased and targets for interventions to improve recovery

A large-volume academic center retrospective audit of the temporal evolution of immediate breast reconstruction protocols and the effect on breast prosthetic infection

Complications of tissue expanders (TEs) in breast reconstruction are challenging. We sought to identify TE infection risks and acellular dermal matrix (ADM) and infection control protocol impacts on infection in a longitudinal study. We retrospectively analyzed TE/implant reconstructions in 2004 (no ADM), 2009 (TE and ADM), 2013 (TE, ADM, and infection control protocol), and 2015 (TE, ADM, and infection control protocol). We assessed demographic, disease, and operative factors and analyzed rates of seroma, hematoma, skin necrosis, and infection. Statistical analysis, including simple and multivariable logistic regression, was performed using Stata v13.1. 478 TEs were placed in 324 women, with a 30% overall patient complication rate (23% of breasts). A total of 14% of TEs became infected. Although unadjusted analysis showed no ADM and infection association (p = 0.269), multivariable logistic regression showed a significant association with more infections (OR: 3.21; 95% CI: 1.13–9.313; p = 0.029). The infection control protocol decreased infections by 28% (16% in 2009 vs 11% in 2013); however, this did not achieve statistical significance (unadjusted p = 0.192, adjusted p = 0.156). Seroma (p < 0.001), older age (p = 0.040), larger mastectomy volume (p = 0.001), smoking (p = 0.037), BMI (p < 0.001), vascular disorders (p = 0.007), and hypertension (p < 0.001) significantly increased infections. Identifiable risks exist in TE/implant breast reconstruction. ADM infection risk may mitigate some potential benefits. Anti-infection protocols may reduce infections, and further investigation may reveal the most effective prophylactic strategies. Absence of major changes in complications over time supports validity of studies examining large numbers of despite evolution of techniques