Rapid, effective, and long-lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves.

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²âº-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²âº-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²âº-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures.

Surgical management and epidemiological trends of pediatric electrical burns.

Electrical burns are an uncommon yet devastating class of burn injuries. Shriners Hospitals for Children - Boston a pediatric burn center in New England and cares for both domestic and international patients. We utilized our experience over the past 13 years to review surgical management and evaluate historical trends for this unique patient group. A retrospective chart review was conducted on 68 patients aged 0-18 years admitted to our pediatric center with an electrical burn from January 2005 to December 2018. We collected and analyzed data pertaining to patient demographics, burn characteristics, clinical course, and surgical interventions. Our cohort included 31 patients from the US (46%) and 37 transferred from a variety of international countries (54%). The majority of US patients were admitted with low voltage burns (81%), whereas the majority of international patients were admitted with high voltage burns (95%). Acute and reconstructive surgical interventions were performed mainly for high voltage burns (94% and 89%). Based on our experience, epidemiology and surgical intervention varied based on voltage of the burn injury and residence of the patient. We have seen a reduction in US pediatric high voltage injuries over the past two decades, likely due to enhancement of electrical safety. It may be possible to use a similar strategy to reduce the frequency of severe high voltage electrical burn injuries in developing countries.

Osteoblastic and osteoclastic activation in coronal sutures undergoing fusion ex vivo.

Numerous studies have demonstrated the importance of dura mater in the normal development and regeneration of the cranium and its sutures. The purpose of this study was to analyze the effect of dura mater on the metabolism of bone during the process of premature suture fusion. Previously, coronal sutures of fetal rats have been shown to fuse in serum-free culture after removal of their dura mater, whereas sutures of neonatal rats resist fusion even without their dura mater present. Sutures from these two distinct developmental stages were evaluated by assaying alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP), marker enzymes of bone synthesis and catabolism, respectively. Coronal sutures with adjacent calvaria were dissected from fetal day 19.5 (F19) rats (n = 142) and neonatal day 1 (N1) rats (n = 42) and randomly divided into two groups each: F19 sutures with dura mater intact; F19 sutures with dura mater removed; N1 sutures with dura mater intact; and N1 sutures with dura mater removed. Calvaria were grown in serum-free medium for up to 21 days, and enzyme activities in suture regions were assayed by microanalytical techniques at different time intervals of culture. F19 sutures without dura mater exhibited significant increases in enzyme activities during days 7 to 21 of culture, whereas those without dura mater did not. N1 sutures with or without dura mater exhibited no significant changes in enzyme activities during the 14-day period of culture. The process of F19 suture fusion, occurring in the absence of dura mater, coincided with the increased activities of both alkaline phosphatase and TRAP. These cellular, enzymatic changes may have implications for the cellular events comprising craniosynostosis in vivo.

Enzymatic activation associated with programmed fusion of the posterior interfrontal sutures in rats.

Fetal rat coronal sutures in culture undergo fusion in the absence of their dura mater. Coinciding with the period of fusion are marked cellular enzymatic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activity, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifically related to bone formation or degradation also show activation within these fusing sutures. These enzymes include tartrate-sensitive acid phosphatase (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monophosphate shunt; and glutathione reductase, an enzyme of the antioxidant pathway. In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the posterior interfrontal suture of the rat. This suture fuses between postnatal days 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a comparable midline suture. Neonatal rats were killed at postnatal days 2, 4, 5, 8, 10, 12, 15, 20, and 30, and posterior interfrontal and sagittal sutures with bone plates on either side were removed. The suture regions of the samples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, TSAP, hexokinase, G6PD, and glutathione reductase were measured. DNA content was also assayed, and enzyme activities were expressed per amount of DNA. Three pups were killed at each time point, and three to five assays were performed per suture (posterior interfrontal or sagittal) for each time point assayed. Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with nonfusing controls, similar to the increases demonstrated ex vivo. TSAP and hexokinase also showed elevations in the fusing posterior interfrontal sutures, with the greatest differences predominantly during the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did not demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples for the majority of time points examined. Alkaline phosphatase and TRAP activity elevations indicated both osteoblastic and osteoclastic activation during fusion, as seen in the ex vivo phenomenon. TSAP and hexokinase increases also reflected activation in lysosomes and in cellular metabolism during fusion, paralleling the ex vivo situation. However, a less clear pattern of activation in the antioxidant pathway, in contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternatively, oxidative stress may play a more central role in the pathologic process of induced suture fusion ex vivo than in programmed suture fusion in vivo.

Successful distraction osteogenesis across a growing cranial suture without an osteotomy.

The application of distraction osteogenesis to the membranous bones of the craniofacial skeleton and its effects on cranial volume and overall skull shape have not been fully studied. This pilot study was designed to compare distraction of a cranial suture in the rabbit model with distraction across an osteotomy and to evaluate the response of the suture both grossly and histologically. Additionally, the need for a period of rigid fixation after distraction was evaluated. Calibrated distraction of either the coronal suture or an osteotomy in the midsection of the parietal bone using an internal distractor (Synthes Maxillofacial, Paoli, Pa.) was studied in juvenile and adult New Zealand White rabbits. Skull growth and distraction were followed by serial cephalograms, and bone biopsies of the distracted sites were obtained after death for histologic evaluation. Craniometric analysis of the skulls was also performed. There was significantly greater mean marker separation in the juvenile suture distraction group (6.6 +/- 0.2 mm; n = 3) than in the control group (2.2 +/- 0.2 mm; p < 0.01). Marker separation was also significantly greater across the parietal osteotomy (6.4 +/- 0.1 mm) than in the sham group (no marker separation). Craniometric data demonstrated the ability of the juvenile skull to distribute the change at the coronal suture throughout the skull to maintain symmetry and minimize disproportion. No distraction was obtained across the adult suture. In the juvenile suture distraction group without a period of stable fixation, a relapse of 17 +/- 6 percent was noted after 1 week, an additional 2.3 +/- 0.5 percent after the second week, and no further change in the third postdistraction week (n = 2; p < 0.01). These results show that in this animal model, distraction osteogenesis can be achieved across a growing cranial suture without an osteotomy or suturectomy and that the degree of distraction and new bone formation is comparable to that across an osteotomy. This suggests that cranial expansion can be obtained in a growing animal without using a more invasive cranial osteotomy. Additionally, the first week after distraction seems to be the most critical time period to maintain stable fixation.

In vivo evaluation of demyelination and remyelination in a nerve crush injury model.

Nerves of the peripheral nervous system have, to some extent, the ability to regenerate after injury, particularly in instances of crush or contusion injuries. After a controlled crush injury of the rat sciatic nerve, demyelination and remyelination are followed with functional assessments and imaged both ex vivo and in vivo over the course of 4 weeks with video-rate coherent anti-Stokes Raman scattering (CARS) microscopy. A new procedure compatible with live animal imaging is developed for performing histomorphometry of myelinated axons. This allows quantification of demyelination proximal and remyelination distal to the crush site ex vivo and in vivo respectively.

Iron-induced rat coronal suture fusion in vitro: the role of redox regulation.

The presumptive coronal sutures of rat fetuses at gestation days 19 and 20 have been shown to fuse prematurely when grown in the absence of dura mater in culture. In the present study, the representative enzymes of glucose metabolism and the antioxidative pathway were assayed during the process of suture fusion. The coronal sutures of fetal day 19.5 (F19) and neonatal day 1 rats were grown in the presence or absence of dura mater in serum-free culture. The enzymes assayed were hexokinase (HK) and pyruvate kinase (PK) of glycolysis, and glucose 6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) of the antioxidative pathway. F19 sutures cultured without dura mater, which fused, showed significant increases in enzyme activities over the preculture levels. HK increased by 200% to 300% of the preculture levels, G6PD by 400% to 500%, GR by 200%, and PK by 400% to 500%. The fetal sutures cultured with dura mater, which did not fuse, showed little alterations of HK, G6PD, and GR activities, but showed a significant 200% to 400% increase in PK activity. Neonatal sutures showed significant increases in enzyme activities during culture, but the presence of dura mater did not significantly affect enzyme activities. High activity levels of enzymes of the antioxidative pathway in F19 sutures coincided with the period of premature suture fusion. Treatment of fetal calvaria with prooxidant (induced by ferrous iron and ascorbic acid) produced suture fusion even in the presence of dura mater. Treatment with deferoxamine (an iron chelator and antioxidant) during the culture prevented suture fusion. The results suggest that fusing sutures experience increased biosynthetic demands and are placed under oxidative stress. When oxidative stress overwhelms the dural influence, the sutures undergo premature fusion.