Antinociceptive effect of vapocoolant medium stream spray on hotplate latency in rat pups.

BACKGROUND: Heel sticks account for most blood tests performed in neonates without analgesia because topical local anesthetics are ineffective on heel glabrous skin. We investigated the antinociceptive effect of an alternative topical analgesic, a vapocoolant spray, on hind paw glabrous skin of rat pups. The spray was applied by two methods: method 1 for 4 s at a distance of 8 cm and method 2 for 10 s at a distance of 18 cm. METHODS: The rat pups were randomized to either method 1 (n = 32) or method 2 (n = 31). Vapocoolant spray was applied to one hind paw randomly, and saline spray was applied to the contralateral paw. The paws were exposed to a hotplate test to measure withdrawal latency time before and 30 s after the spray applications. Additionally, rat pups were tested for tissue toxicity in method 1 (n = 20) and method 2 (n = 20) after application of the vapocoolant spray before heel sticks three times a day for two consecutive days. Analyses of spray and method effects on hotplate withdrawal latency time were determined by nonparametric Wilcoxon tests to assess paired difference between vapocoolant spray and saline spray and to compare difference in medians between the two methods. RESULTS: Method 1 and method 2 vapocoolant spray applications significantly prolonged the withdrawal latency time compared with saline, a median difference of 0.6 s (IQR 0.1-1.2) for method 1 and 9.5 s (IQR 5.5-10.7) for method 2 (a 15-fold longer latency time with method 2). Method-2 produced significantly longer withdrawal latency time than method 1 with a difference in median time of 8.9 s (CI: 95% 7.3-10.4 s, P < .0001). No histopathological changes were detected. CONCLUSIONS: Compared with method- 1, the vapocoolant spray in method 2 produced significantly longer withdrawal latency time that is clinically applicable to collecting blood samples after a heel stick.

Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot System as Compared to Enzyme Replacement Therapy.

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-galactosidase A. Enzyme deficiency results in a progressive decline in renal and cardiac function, leading to cardiomyopathy and end-stage renal disease. Current treatments available, including enzyme replacement therapies, have provided significant benefit to patients; however, unmet medical needs remain. mRNA therapy, with drug-like properties, has the unique ability to produce therapeutic proteins endogenously. Here we describe the sustained delivery of therapeutic human α-galactosidase protein in vivo via nanoparticle-formulated mRNA in mouse and non-human primate, with a demonstration of efficacy through clinically relevant biomarker reduction in a mouse Fabry disease model. Multi-component nanoparticles formulated with lipids and lipid-like materials were developed for the delivery of mRNA encoding human α-galactosidase protein. Upon delivery of human GLA mRNA to mice, serum GLA protein levels reached as high as ∼1,330-fold over normal physiological values.

The analgesic effect of a vapocoolant stream spray in reducing heat nociception on the glabrous skin of rat pups.

BACKGROUND: Blood sampling is a common screening and diagnostic test in newborn infants in the neonatal intensive care unit, and heel lancing accounts for two-thirds of these tests. Heel lancing causes acute pain and distress, and most infants rarely receive analgesics because of fear of respiratory depression from opioids and lack of effectiveness of topical local anesthetics on the glabrous skin. To circumvent this latter problem, we investigated the analgesic efficacy and safety of a topical vapocoolant spray. METHODS: Forty Sprague-Dawley rat pups aged 7 days old were randomly assigned to receive either vapocoolant or saline spray on the plantar hindpaws for 5 to 6 seconds. Forty-five seconds later, the paws were subjected to a modified hotplate test to quantify the nociceptive flexor withdrawal (NFW) thresholds before and after treatment with the sprays. Seven days later, the animals were euthanized and the hindpaws were examined histologically. A nested analysis of variance approach was used to account for the triplicate measurements per animal. A 2-tailed P < 0.05 was considered significant. RESULTS: At baseline, there were no differences in the NFW thresholds between the 2 groups (P = 0.22). After treatment, these thresholds were significantly lower in both vapocoolant (P < 0.001) and saline (P = 0.008) groups relative to baseline values. The vapocoolant group demonstrated a significantly longer NFW latency time compared to the saline group (P < 0.001). All specimens in both groups were examined and showed normal skin histology. CONCLUSIONS: Vapocoolant spray treatment of the glabrous skin is effective and safe after a single treatment.

Development of standardized insulin treatment protocols for spontaneous rodent models of type 1 diabetes.

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.

Prevention of type 1 diabetes in the rat with an allele-specific anti-T-cell receptor antibody: Vß13 as a therapeutic target and biomarker.

In earlier studies of the Iddm14 diabetes susceptibility locus in the rat, we identified an allele of the T-cell receptor (TCR) ß-chain, Tcrb-V13S1A1, as a candidate gene. To establish its importance, we treated susceptible rats with a depleting anti-rat Vß13 monoclonal antibody and then exposed them to either polyinosinic:polycytidylic acid or a diabetogenic virus to induce diabetes. The overall frequency of diabetes in the controls was 74% (n = 50), compared with 17% (n = 30) in the anti-Vß13-treated animals, with minimal islet pathology in nondiabetic treated animals. T cells isolated from islets on day 5 after starting induction showed a greater proportion of Vß13(+) T cells than did peripheral lymph node T cells. Vß13 transcripts recovered from day 5 islets revealed focused Jß usage and less CDR3 diversity than did transcripts from peripheral Vß13(+) T cells. CDR3 usage was not skewed in control Vß16 CDR3 transcripts. Anti-rat Vß13 antibody also prevented spontaneous diabetes in BBDP rats. The Iddm14 gene is likely to be Tcrb-V13, indicating that TCR ß-chain usage is a determinant of susceptibility to autoimmune diabetes in rats. It may be possible to prevent autoimmune diabetes by targeting a limited element of the T-cell repertoire.

Spinal anesthesia in infant rats: development of a model and assessment of neurologic outcomes.

BACKGROUND: Previous studies in infant rats and case-control studies of human infants undergoing surgery have raised concerns about potential neurodevelopmental toxicities of general anesthesia. Spinal anesthesia is an alternative to general anesthesia for some infant surgeries. To test for potential toxicity, a spinal anesthesia model in infant rats was developed. METHODS: Rats of postnatal ages 7, 14, and 21 days were assigned to no treatment, 1% isoflurane for either 1 h or 6 h, or lumbar spinal injection of saline or bupivacaine at doses of 3.75 mg/kg (low dose) or 7.5 mg/kg (high dose). Subgroups of animals underwent neurobehavioral testing and blood gas analysis. Brain and lumbar spinal cord sections were examined for apoptosis using cleaved caspase-3 immunostaining. The lumbar spinal cord was examined histologically.Rats exposed to spinal or general anesthesia as infants underwent Rotarod testing of motor performance as adults. Data were analyzed using ANOVA with general linear models, Friedman tests, and Mann-Whitney U tests, as appropriate. RESULTS: Bupivacaine 3.75 mg/kg was effective for spinal anesthesia in all age groups. Impairments in sensory and motor function recovered in 40-60 min. Blood gases were similar among groups. Brain and spinal cord apoptosis increased in rats receiving 6 h of 1% isoflurane, but not among the other treatments. All groups showed intact motor performance at adulthood. CONCLUSIONS: Spinal anesthesia is technically feasible in infant rats and appears benign in terms of neuroapoptotic and neuromotor sequelae.

Tetrodotoxin-bupivacaine-epinephrine combinations for prolonged local anesthesia.

Currently available local anesthetics have analgesic durations in humans generally less than 12 hours. Prolonged-duration local anesthetics will be useful for postoperative analgesia. Previous studies showed that in rats, combinations of tetrodotoxin (TTX) with bupivacaine had supra-additive effects on sciatic block durations. In those studies, epinephrine combined with TTX prolonged blocks more than 10-fold, while reducing systemic toxicity. TTX, formulated as Tectin, is in phase III clinical trials as an injectable systemic analgesic for chronic cancer pain. Here, we examine dose-duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine 0.25% (2.5 mg/mL) solutions, with or without epinephrine 5 µg/mL (1:200,000) in rats. Percutaneous sciatic blockade was performed in Sprague-Dawley rats, and intensity and duration of sensory blockade was tested blindly with different Tectin-bupivacaine-epinephrine combinations. Between-group comparisons were analyzed using ANOVA and post-hoc Sidak tests. Nerves were examined blindly for signs of injury. Blocks containing bupivacaine 0.25% with Tectin 10 µM and epinephrine 5 µg/mL were prolonged by roughly 3-fold compared to blocks with bupivacaine 0.25% plain (P < 0.001) or bupivacaine 0.25% with epinephrine 5 µg/mL (P < 0.001). Nerve histology was benign for all groups. Combinations of Tectin in bupivacaine 0.25% with epinephrine 5 µg/mL appear promising for prolonged duration of local anesthesia.