Molecular and cellular correlates of human nerve regeneration: ADCYAP1/PACAP enhance nerve outgrowth

We only have a rudimentary understanding of the molecular and cellular determinants of nerve regeneration and neuropathic pain in humans. This cohort study uses the most common entrapment neuropathy (carpal tunnel syndrome) as a human model system to prospectively evaluate the cellular and molecular correlates of neural regeneration and its relationship with clinical recovery. In 60 patients undergoing carpal tunnel surgery [36 female, mean age 62.5 (standard deviation 12.2) years], we used quantitative sensory testing and nerve conduction studies to evaluate the function of large and small fibres before and 6 months after surgery. Clinical recovery was assessed with the global rating of change scale and Boston Carpal Tunnel Questionnaire. Twenty healthy participants provided normative data [14 female, mean age 58.0 (standard deviation 12.9) years]. At 6 months post-surgery, we noted significant recovery of median nerve neurophysiological parameters (P < 0.0001) and improvements in quantitative sensory testing measures of both small and large nerve fibre function (P < 0.002). Serial biopsies revealed a partial recovery of intraepidermal nerve fibre density [fibres/mm epidermis pre: 4.20 (2.83), post: 5.35 (3.34), P = 0.001], whose extent correlated with symptom improvement (r = 0.389, P = 0.001). In myelinated afferents, nodal length increased postoperatively [pre: 2.03 (0.82), post: 3.03 (1.23), P < 0.0001] suggesting that this is an adaptive phenomenon. Transcriptional profiling of the skin revealed 31 differentially expressed genes following decompression, with ADCYAP1 (encoding pituitary adenylate cyclase activating peptide, PACAP) being the most strongly upregulated (log2 fold-change 1.87, P = 0.0001) and its expression was associated with recovery of intraepidermal nerve fibres. We found that human induced pluripotent stem cell-derived sensory neurons expressed the receptor for PACAP and that this peptide could significantly enhance axon outgrowth in a dose-dependent manner in vitro [neurite length PACAP 1065.0 µm (285.5), vehicle 570.9 μm (181.8), P = 0.003]. In conclusion, carpal tunnel release is associated with significant cutaneous reinnervation, which correlates with the degree of functional improvement and is associated with a transcriptional programme relating to morphogenesis and inflammatory processes. The most highly dysregulated gene ADCYAP1 (encoding PACAP) was associated with reinnervation and, given that this peptide signals through G-protein coupled receptors, this signalling pathway provides an interesting therapeutic target for human sensory nerve regeneration

Validity of clinical small-fiber sensory testing to detect small-nerve fiber degeneration

Study Design Prospective, cross-sectional diagnostic accuracy study. Background Small nerve fiber degeneration (SFD) commonly occurs in patients with peripheral neuropathies, resulting in deterioration in nerve function. Currently, the gold standard to identify SFD is through skin biopsies. Simple clinical tests aim to identify SFD, but their validity remains unknown. Objectives To examine the validity of clinical tests to assess SFD using carpal tunnel syndrome (CTS) as a model system. Methods 107 participants (22 healthy, 85 with CTS) underwent pinprick testing over the index finger, and had cold and warm detection thresholds taken (CDT, WDT) using quantitative sensory testing (QST). In a sub-group of patients with CTS (n=51), cold and warm sensation were also tested using coins at room and body temperature respectively. The validity of these clinical tests was established against intra-epidermal nerve fiber density (IENFD) measured in skin biopsies from the index finger. Results Optimal validity occurred with clusters of tests: normal warm OR cold sensation is highly sensitive to rule out SFD (sensitivity 0.98, 95% CI 0.85,0.99), but has a low specificity (0.2, 95% CI 0.03,0.52). A reduction in pinprick in contrast is highly specific (0.88, 95% CI 0.72,0.95), thus useful to rule in SFD. For QST, highest specificity (0.77) occurred for WDT and highest sensitivity (0.84, 95% CI, 0.72,0.91) for CDT OR WDT. Conclusion Pin prick testing followed by warm and cold tests if pin prick is normal, is a valid and cost-effective method to detect SFD. For QST, WDT is useful for ruling in SFD. To rule out SFD both CDT and WDT must be negative

Higher densities of T-lymphocytes in the subsynovial connective tissue of people with carpal tunnel syndrome.

Symptoms in people with carpal tunnel syndrome (CTS) are traditionally attributed to neural tissue, but recent studies suggest that the subsynovial connective tissue (SSCT) may also play a role in CTS. The SSCT undergoes fibrotic thickening which is generally described as "non-inflammatory" based on basic histology. This study uses immunohistochemistry to determine the presence of macrophages and T-cells within SSCT and their relationship with symptoms in people with CTS. SSCT was collected from twenty people with CTS and eight controls undergoing wrist fracture surgery. Immunohistochemical quantification of CD3+ T-cells and CD68+ macrophage densities as well as CD4+/CD8+ T-cell subpopulations were compared between groups using independent t-tests. Spearman correlations were used to identify associations between immune cell densities and CTS symptom scores. The density of CD3+ T-cells was significantly higher in SSCT of people with CTS compared to controls (CTS mean 26.7 (SD 13.7); controls 6.78 (6.3), p = 0.0005) while the density of CD68+ macrophages was lower (CTS mean 9.5 (SD 6.0); controls 17.7 (8.2), p = 0.0058). Neither CD68+ nor CD3+ cell densities correlated with symptom scores. In contrast to previous assumptions, our data show that the SSCT in the carpal tunnel in both people with CTS and controls is not devoid of immune cells. Whereas the higher density of CD68+ macrophages in control participants may be associated with their early recruitment after acute fracture, CD3+ cells within the SSCT may play a role in chronic CTS

No association between PGP+ IENF regeneration and the percentage of PGP+IENF containing GAP-43.

Graph shows pre surgical (green) and post-surgical (blue) PGP+ IENF containing GAP-43 with cubic spline curves showing no association. (TIFF)</p

Participant characteristics of patients classified as regenerators and non-regenerators.

Participant characteristics of patients classified as regenerators and non-regenerators.</p

GAP-43 Gene expression.

No difference in GAP-43 gene expression in skin of patients with CTS before and after carpal tunnel decompression. Normalised log2 counts of (A) Gap-43 and (B) GAP-43 gene expression expressed as a percentage of PGP9.5 gene expression are comparable before and after carpal tunnel surgery. No differences in (C) GAP-43 gene expression and (D) GAP-43 expressed as a percentage of PGP9.5 gene expression in skin of patients classified as regenerators and non-regenerators both before and after surgery.</p

Median nerve neurophysiology data of healthy participants and patients with CTS pre- and post-surgery.

Data are presented as median [interquartile range]. (DOCX)</p

Immunohistochemical staining results of GAP-43 and PGP.

(A) Patients with CTS before surgery have fewer GAP-43+ IENF than healthy controls. No changes in GAP-43+ IENFD was apparent from before to six months after surgery in patients with CTS. (B) No difference was apparent in the % of PGP+ IENF containing GAP-43 among healthy participants, patients with CTS before and after surgery. (C) IENFD of patients with CTS classified as regenerators and non-regenerators according to the difference in PGP+ IENFD before and after carpal tunnel surgery. (D) No difference was apparent in the percentage of PGP+ IENF containing GAP-43 between regenerators and non-regenerators both pre- and post-surgery. *p = 0.036.</p

Participant characteristics.

Participant characteristics.</p

Spearman correlations between pre and post-operative cutaneous GAP-43 expression (histological analysis) and pain characteristics according to the Neuropathic Pain Symptom Inventory.

Table shows p-values. No significant correlations were identified. (DOCX)</p