Carla Porretta-Serapiglia

Morphometry of dermal nerve fibers in human skin

Objective: We aimed to assess the innervation density of dermal nerves in human skin biopsies by bright-field immunohistochemistry. Methods: The size of dermal area where nerve length was quantified was validated in 30 skin biopsy sections (5 controls and 5 patients with small-fiber neuropathy [SFN]). It was obtained dividing an area of 200-μm depth from the dermal-epidermal junction into 4 equal portions. The length of dermal nerves (DNFL) was measured into 150 sections (25 controls and 25 patients with SFN) and values per millimeter of epidermis (DNFL/mm) and dermal area (DNFL/mm2) were obtained. Age- and gender-matched normative values of intraepidermal nerve fiber (IENF) density were used as gold standard to calculate the performance of dermal nerve morphometry. Results: Patients showed significantly lower DNFL (1.96 mm ± 0.96 SD), DNFL/mm (0.65 ± 0.29 SD), and DNFL/mm2 (3.75 ± 1.7 SD) than controls (DNFL 3.52 mm ± 1.31 SD, 5th percentile 2.05; DNFL/mm 1.25 ± 0.39, 5th percentile 0.71; DNFL/mm2 7.07 ± 2.41 SD, 5th percentile 3.95). Sensitivity, specificity, and percentage of individuals correctly classified were 75.8%, 73.9%, and 74.8% for DNFL, 75%, 80%, and 77.7% for DNFL/mm, and 75.8%, 80.2%, and 78.1% for DNFL/mm2. Receiver operator characteristic area analysis confirmed the excellent discrimination (0.8–0.9) between patients and controls. Dermal nerve morphometry significantly correlated with IENF density. Spearman rank correlation demonstrated good agreement for interobserver analysis (0.87–0.89), and between DNFL and IENF densities (0.71–0.73; p < 0.0001). Conclusions: We provided a reliable method to quantify the innervation density of dermal nerves that might improve the diagnostic yield of skin biopsy.

Beneficial Effects of PKF275-055, a Novel, Selective, Orally Bioavailable, Long-Acting Dipeptidyl Peptidase IV Inhibitor in Streptozotocin-Induced Diabetic Peripheral Neuropathy

1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275–055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na+/K+-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275-055 induced a significant correction in the alteration in Na+,K+-ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na+,K+-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.

Amyotrophic lateral sclerosis causes small fiber pathology

Our aim was to address the correlation between small fiber loss and amyotrophic lateral sclerosis (ALS) for disease onset, phenotype, genotype, duration, severity and sensory findings.

Hydroxytyrosol attenuates peripheral neuropathy in streptozotocin-induced diabetes in rats

Peripheral neuropathy is one of the most frequent and severe complications of diabetes. Hydroxytyrosol (HT), the major antioxidant polyphenolic compound of olive oil, has been investigated as a new potential treatment to counteract the progression of peripheral diabetic neuropathy in rats. An established model of streptozotocin-induced diabetes has been used. After confirmation of hyperglycemia, diabetic and nondiabetic animals were randomized to receive either a low dose or a high dose of HT, or the corresponding vehicle, for 6 weeks. At the end of the 6-week period of treatment, HT blunted plasma thiobarbituric acid-reactive substances increase (p < 0.05) and significantly reduced nerve conduction velocity (p < 0.05) and thermal nociception impairment in diabetic rats (p < 0.05). Sciatic nerve Na(+), K(+)-ATPase activity reduction was also abolished by HT (p < 0.05). The present study provides evidence of the therapeutic potential of the natural substance hydroxytyrosol in the early stage of diabetic neuropathy.

Lowering Plasma 1-Deoxysphingolipids Improves Neuropathy in Diabetic Rats

1-Deoxysphingolipids (1-deoxySLs) are atypical neurotoxic sphingolipids that are formed by the serine-palmitoyltransferase (SPT). Pathologically elevated 1-deoxySL concentrations cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonal neuropathy associated with several missense mutations in SPT. Oral L-serine supplementation suppressed the formation of 1-deoxySLs in patients with HSAN1 and preserved nerve function in an HSAN1 mouse model. Because 1-deoxySLs also are elevated in patients with type 2 diabetes mellitus, L-serine supplementation could also be a therapeutic option for diabetic neuropathy (DN). This was tested in diabetic STZ rats in a preventive and therapeutic treatment scheme. Diabetic rats showed significantly increased plasma 1-deoxySL concentrations, and L-serine supplementation lowered 1-deoxySL concentrations in both treatment schemes (P < 0.0001). L-serine had no significant effect on hyperglycemia, body weight, or food intake. Mechanical sensitivity was significantly improved in the preventive (P < 0.01) and therapeutic schemes (P < 0.001). Nerve conduction velocity (NCV) significantly improved in only the preventive group (P < 0.05). Overall NCV showed a highly significant (P = 5.2E-12) inverse correlation with plasma 1-deoxySL concentrations. In summary, our data support the hypothesis that 1-deoxySLs are involved in the pathology of DN and that an oral L-serine supplementation could be a novel therapeutic option for treating DN.

Diabetic neuropathic pain: a role for testosterone metabolites

Diabetic neuropathy is associated with neuropathic pain in about 50% of diabetic subjects. Clinical management of neuropathic pain is complex and so far unsatisfactory. In this study, we analyzed the effects of the testosterone metabolites, dihydrotestosterone (DHT), and 3α-diol, on nociceptive and allodynia thresholds and on molecular and functional parameters related to pain modulation in the dorsal horns of the spinal cord and in the dorsal root ganglia of rats rendered diabetic by streptozotocin injection. Furthermore, the levels of DHT and 3α-diol were analyzed in the spinal cord. Diabetes resulted in a significant decrease in DHT levels in the spinal cord that was reverted by DHT or 3α-diol treatments. In addition, 3α-diol treatment resulted in a significant increase in 3α-diol in the spinal cord compared with control values. Both steroids showed analgesic properties on diabetic neuropathic pain, affecting different pain parameters and possibly by different mechanisms of action. Indeed, DHT counteracted the effect of diabetes on the mechanical nociceptive threshold, pre- and post-synaptic components, glutamate release, astrocyte immunoreactivity, and expression of interleukin-1β (IL1β), while 3α-diol was effective on tactile allodynia threshold, glutamate release, astrocyte immunoreactivity and the expression of substance P, toll-like receptor 4, tumor necrosis factor-α, transforming growth factor β-1, IL1β, and translocator protein. These results indicate that testosterone metabolites are potential agents for the treatment of diabetic neuropathic pain.

Continuous Buprenorphine Delivery Effect in Streptozotocine-Induced Painful Diabetic Neuropathy in Rats

UNLABELLED Diabetic peripheral neuropathy (DPN) can induce loss of nociception as well as mechanical hyperalgesia and tactile allodynia. Pharmacological and clinical studies have shown that buprenorphine, a low-molecular-weight, lipophilic, opioid analgesic available as a transdermal matrix patch formulation, acts on neuropathic pain. To assess the role of buprenorphine in the treatment of DPN-associated neuropathic pain, we used a well-established experimental rat model of DPN in which buprenorphine at doses of 1.2 and 2.4 microg/kg/h was administered by implantable Alzet osmotic pumps for 3 weeks. After 6 weeks of diabetes, nerve conduction velocity (NCV) and behavioural responses to noxious mechanical and thermal stimuli were assessed. Diabetic rats showed an impairment of NCV, mechanical allodynia, and thermal hypoalgesia. Both doses of buprenorphine significantly reversed the diabetes-induced allodynia up to day 7 of treatment. Buprenorphine did not alter either thermal perception or NCV. PERSPECTIVE This study evaluated, through a multimodal approach, the analgesic effect of buprenorphine in an experimental rat model of painful DPN. Our results suggest a possible role for buprenorphine in the management of DPN-associated neuropathic pain.

Lack of sterol regulatory element binding factor-1c imposes glial fatty acid utilization leading to peripheral neuropathy

Myelin is a membrane characterized by high lipid content to facilitate impulse propagation. Changes in myelin fatty acid (FA) composition have been associated with peripheral neuropathy, but the specific role of peripheral nerve FA synthesis in myelin formation and function is poorly understood. We have found that mice lacking sterol regulatory element-binding factor-1c (Srebf1c) have blunted peripheral nerve FA synthesis that results in development of peripheral neuropathy. Srebf1c-null mice develop Remak bundle alterations and hypermyelination of small-caliber fibers that impair nerve function. Peripheral nerves lacking Srebf1c show decreased FA synthesis and glycolytic flux, but increased FA catabolism and mitochondrial function. These metabolic alterations are the result of local accumulation of two endogenous peroxisome proliferator-activated receptor-α (Pparα) ligands, 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine and 1-stearoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine. Treatment with a Pparα antagonist rescues the neuropathy of Srebf1c-null mice. These findings reveal the importance of peripheral nerve FA synthesis to sustain myelin structure and function.

Epidermal innervation morphometry by immunofluorescence and bright-field microscopy

We investigated the agreement between simple indirect immunofluorescence (IF) and bright‐field immunohistochemistry (BFI) on free‐floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty‐five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland–Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age‐adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland–Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut‐off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values.

Dermal innervation in healthy subjects and small fiber neuropathy patients: a stereological reappraisal.

The aim of this study was to estimate dermal nerve fiber length (DNFL) using a stereological sampling technique in comparison with a previously reported manual estimation. DNFL was analyzed in skin punch biopsy specimens from 24 healthy volunteers and 18 patients with small fiber neuropathy (SFN) using global spatial sampling that yields unbiased and reliable length estimation. The estimation was carried out in 50‐µm biopsy sections after immunostaining with anti‐protein gene product (PGP) 9.5 antibodies. The length of the PGP9.5‐positive dermal nerves from the dermal–epidermal junction and 200 µm down was measured (DNFL mm−2). Results were compared with our previously reported manual method. Patients showed a significantly (p < 0.0001) lower DNFL (105 mm−2 ± 6.4 SD) than healthy subjects (246 mm−2 ± 8.39 SD). Moderate correlation with age was observed for both healthy subjects (Pearsons r = −0.33) and patients (Pearsons r = −0.59). A significant (p < 0.001) correlation between global spatial sampling and manual estimation was observed in both patients and healthy subjects (Pearsons r = 0.62 and 0.61, respectively). These findings provide further evidence on the reliability of dermal nerve morphometry in human skin and strengthen the hypothesis that dermal nerve fibers undergo significant degeneration in SFN.