Increasing Calcium Level Limits Schwann Cell Numbers In Vitro following Peripheral Nerve Injury.

Background After peripheral nerve injury, there is an increase in calcium concentration in the injured nerves. Our previous publications have shown that increase in calcium concentration correlated well with degree of nerve injury and that local infusion of calcitonin has a beneficial effect on nerve recovery. Schwann cells play a pivotal role in regeneration and recovery. We aim to examine cultured Schwann cell survivals in various concentrations of calcium-containing growth media and the effect of calcitonin in such media. Methods To establish baseline in postinjury state, crush injury was induced in male Sprague-Dawley rats' sciatic nerves. Extra- and intraneural calcium concentrations were measured. To study Schwann cell survival, uninjured sciatic nerve segment was harvested and cultured in media containing various amounts of calcium. To study the effect of calcitonin, nerve harvest and culture were done in four additional media: (1) normal control, (2) normal control with calcitonin, (3) high calcium medium, and (4) high calcium medium with calcitonin. Schwann cells were studied and analyzed under fluorescent conditions. Results With increasing calcium concentration, there was a significant decrease in the number of Schwann cells. For the experimental groups, in which calcitonin had been added to the growth medium, there were similar amounts of Schwann cells present in both high and low calcium-containing medium. Conclusion Schwann cells are sensitive to increasing calcium concentration. Calcitonin counteracts the detrimental effects of high calcium on Schwann cell survival. This can have significant future clinical implications for patients with peripheral nerve injuries.

Best time window for the use of calcium-modulating agents to improve functional recovery in injured peripheral nerves-An experiment in rats.

Peripheral nerve injury can have a devastating effect on daily life. Calcium concentrations in nerve fibers drastically increase after nerve injury, and this activates downstream processes leading to neuron death. Our previous studies showed that calcium-modulating agents decrease calcium accumulation, which aids in regeneration of injured peripheral nerves; however, the optimal therapeutic window for this application has not yet been identified. In this study, we show that calcium clearance after nerve injury is positively correlated with functional recovery in rats suffering from a crushed sciatic nerve injury. After the nerve injury, calcium accumulation increased. Peak volume is from 2 to 8 weeks post injury; calcium accumulation then gradually decreased over the following 24-week period. The compound muscle action potential (CMAP) measurement from the extensor digitorum longus muscle recovered to nearly normal levels in 24 weeks. Simultaneously, real-time polymerase chain reaction results showed that upregulation of calcium-ATPase (a membrane protein that transports calcium out of nerve fibers) mRNA peaked at 12 weeks. These results suggest that without intervention, the peak in calcium-ATPase mRNA expression in the injured nerve occurs after the peak in calcium accumulation, and CMAP recovery continues beyond 24 weeks. Immediately using calcium-modulating agents after crushed nerve injury improved functional recovery. These studies suggest that a crucial time frame in which to initiate effective clinical approaches to accelerate calcium clearance and nerve regeneration would be prior to 2 weeks post injury. © 2017 Wiley Periodicals, Inc.

Effect of calcitonin on cultured schwann cells.

INTRODUCTION: After nerve injury, calcium concentrations in intranerve fibers quickly increase. We have shown that functional recovery of injured nerves correlates with calcium absorption. A slight increase in calcium reduces the number of Schwann cells present. Calcitonin therapy greatly improves regeneration by accelerating calcium absorption. We examined the effect of adding calcitonin to higher concentration calcium media on cultured Schwann cells. METHODS: The cells, isolated from intact sciatic nerves, were cultured with normal or higher concentration calcium media with or without calcitonin. Schwann cells were incubated with anti-S-100, goat-anti-mouse, and propidium iodide and then viewed through fluorescent light and phase-contrast microscopy for observation and analysis. RESULTS: The cells in each calcitonin-containing medium showed many Schwann cells, however, the cells in the higher concentration calcium media showed fewer and more defective Schwann cells. CONCLUSION: These results show that calcitonin protects against the harmful effects of excessive calcium encountered in peripheral nerve injury. Muscle Nerve 56: 768-772, 2017.

Early Axonal Area Measurement Predicts Early Nerve Regeneration Outcomes.

BACKGROUND: Study of peripheral nerve injury and regeneration in laboratory animals can be time consuming and expensive. This study determines if it is possible to reduce time and cost for a peripheral nerve regeneration study. PURPOSE: The purpose of this study was to determine if nerve axonal area (NXA) or nerve fiber counting (NFC) correlates with compound muscle action potential (CMAP) recovery which is known to predict functional muscular recovery in the early stage of nerve regeneration. METHODS: In this study, six rats had a crush injury of the sciatic nerve without treatment. These rats were evaluated at 4 weeks of recovery with the following assessments: CMAP readings from the extensor digitorum longus, NXA measurement, and NFC. RESULTS: NXA correlated with CMAP; NFC did not correlate with CMAP. CONCLUSION: NFC is not a reliable method for predicting muscular recovery in the early stages. NXA is a dependable assessment for muscular recovery in the early stages of nerve regeneration. Using NXA measurement can predict later electrophysiological and functional recovery. Using NXA with CMAP measurement for nerve injury, repair, and treatment in the animal study can save cost and time.

Cumulative Brain Injury from Motor Vehicle-Induced Whole-Body Vibration and Prevention by Human Apolipoprotein A-I Molecule Mimetic (4F) Peptide (an Apo A-I Mimetic).

BACKGROUND: Insidious cumulative brain injury from motor vehicle-induced whole-body vibration (MV-WBV) has not yet been studied. The objective of the present study is to validate whether whole-body vibration for long periods causes cumulative brain injury and impairment of the cerebral function. We also explored a preventive method for MV-WBV injury. METHODS: A study simulating whole-body vibration was conducted in 72 male Sprague-Dawley rats divided into 9 groups (N = 8): (1) 2-week normal control; (2) 2-week sham control (in the tube without vibration); (3) 2-week vibration (exposed to whole-body vibration at 30 Hz and .5 G acceleration for 4 hours/day, 5 days/week for 2 weeks; vibration parameters in the present study are similar to the most common driving conditions); (4) 4-week sham control; (5) 4-week vibration; (6) 4-week vibration with human apolipoprotein A-I molecule mimetic (4F)-preconditioning; (7) 8-week sham control; (8) 8-week vibration; and (9) 8-week 4F-preconditioning group. All the rats were evaluated by behavioral, physiological, and histological studies of the brain. RESULTS: Brain injury from vibration is a cumulative process starting with cerebral vasoconstriction, squeezing of the endothelial cells, increased free radicals, decreased nitric oxide, insufficient blood supply to the brain, and repeated reperfusion injury to brain neurons. In the 8-week vibration group, which indicated chronic brain edema, shrunken neuron numbers increased and whole neurons atrophied, which strongly correlated with neural functional impairment. There was no prominent brain neuronal injury in the 4F groups. CONCLUSIONS: The present study demonstrated cumulative brain injury from MV-WBV and validated the preventive effects of 4F preconditioning.

Comparison of Peripheral Nerve Axonal Area Differences in Central and Peripheral Zones of Injured and Repaired Nerves.

BACKGROUND: Histological analysis remains a cornerstone approach for the investigation of peripheral nerve regeneration. This study investigates a newly recognized histological difference between peripheral and central zones within the regenerating nerve trunks. PURPOSE: The purpose of the study was to determine if the nerve axonal area (NXA) in regenerating peripheral nerves differs within central and peripheral areas, when viewed in cross-section. METHODS: A total of 14 rats were divided into two groups, and subjected to different injuries to the right sciatic nerve. Group 1: Transection injury with immediate repair. Group 2: Crush injury without any treatment. The left sciatic nerve was left uninjured and served as a control in each rat. Following 4 weeks of recovery, nerve trunk cross-sections were prepared. Computerized techniques were then employed to divide nerve sections into central and peripheral zones and calculate corresponding NXA values for subsequent statistical analysis. RESULTS: NXA of injured nerves was greater within peripheral as compared with the central zones, independent of injury type (p < 0.05). No statistically significant difference existed within the control groups or between the injury methods with regards to NXA regeneration extent. CONCLUSION: NXA in regenerating peripheral nerves was greater in the peripheral zones than within the central zones.

Neural systemic impairment from whole-body vibration.

Insidious brain microinjury from motor vehicle-induced whole-body vibration (WBV) has not yet been investigated. For a long time we have believed that WBV would cause cumulative brain microinjury and impair cerebral function, which suggests an important risk factor for motor vehicle accidents and secondary cerebral vascular diseases. Fifty-six Sprague-Dawley rats were divided into seven groups (n = 8): 1) 2-week normal control group, 2) 2-week sham control group (restrained in the tube without vibration), 3) 2-week vibration group (exposed to whole-body vibration at 30 Hz and 0.5g acceleration for 4 hr/day, 5 days/week, for 2 weeks), 4) 4-week sham control group, 5) 4-week vibration group, 6) 8-week sham control group, and 7) 8-week vibration group. At the end point, all rats were evaluated in behavior, physiological, and brain histopathological studies. The cerebral injury from WBV is a cumulative process starting with vasospasm squeezing of the endothelial cells, followed by constriction of the cerebral arteries. After the 4-week vibration, brain neuron apoptosis started. After the 8-week vibration, vacuoles increased further in the brain arteries. Brain capillary walls thickened, mean neuron size was obviously reduced, neuron necrosis became prominent, and wide-ranging chronic cerebral edema was seen. These pathological findings are strongly correlated with neural functional impairments.

Calcitonin pump improves nerve regeneration after transection injury and repair.

INTRODUCTION: After nerve injury, excessive calcium impedes nerve regeneration. We previously showed that calcitonin improved nerve regeneration in crush injury. We aimed to validate the direct effect of calcitonin on transected and repaired nerve. METHODS: Two rat groups (n = 8) underwent sciatic nerve transection followed by direct repair. In the calcitonin group, a calcitonin-filled mini-osmotic pump was implanted subcutaneously, with a catheter parallel to the repaired nerve. The control group underwent repair only, without a pump. Evaluation and comparison between the groups included: (1) compound muscle action potential recording of the extensor digitorum longus (EDL) muscle; (2) tetanic muscle force test of EDL; (3) nerve calcium concentration; and (4) nerve fiber count and calcified spot count. RESULTS: The calcitonin pump group showed superior recovery. CONCLUSIONS: Calcitonin affects injured and repaired peripheral nerve directly. The calcitonin-filled mini-osmotic pump improved nerve functional recovery by accelerating calcium absorption from the repaired nerve. This finding has potential clinical applications.

A quantitative study of vibration injury to peripheral nerves-introducing a new longitudinal section analysis.

PURPOSE: Long-term vibrations are known to cause neurovascular diseases, which are common in workers who operate handheld power tools or motor vehicles. Understanding the neuropathology of vibration-induced nerve injury is critical to its prevention and treatment. This study aims to evaluate whether light microscopy of longitudinal nerve sections can be used as a simple yet effective method for quantifying nerve injury. METHODS: The rats were split into two groups that were subjected to vibration (4 h/day) for 7 or 14 days. They were then allowed to rest for varying periods of time. Longitudinal sections of the tail nerves were examined under light microscopy. Injuries to the nerves were classified into three types, counted, tallied, and then divided by the length of the nerve being studied. RESULTS: Both 7 and 14 days of vibration showed significant damage when no recovery time was given. After 1 month of rest, the 7-day group began to show signs of recovery, but the 14-day group did not. After 2 months of rest, the 7-day vibration group showed almost complete recovery, while the 14-day vibration group still showed significant damage when compared to the sham control groups. CONCLUSION: The amount of damage to the myelin sheath directly correlated with vibration duration. When vibrated for longer than 7 days, nerve recovery was limited. This study also demonstrated that light microscopy of longitudinal slices is a simple yet effective method of quantifying the nerve damage.

A new computerized morphometric analysis for peripheral nerve study.

The commonly used methods to quantify axon numbers and mean area include manual and semiautomated procedures. The authors introduce a new fully automated method of morphometric analysis using ImageJ and Paint.net software to improve efficiency and accuracy. A total of six rat sciatic nerves were examined for their axon numbers and mean axon area by comparing the manual method or semiautomated MetaVue method with the new ImageJ method. It was observed that the number of axons for manual counting and ImageJ were 4,630 ± 403 and 4,779 ± 352, respectively, and the difference was not statistically significant (p > 0.5, t-test). The mean axon area measured was 13.44 ± 2.62 µm2 for MetaVue and 8.87 ± 0.78 µm2 for ImageJ, respectively, and the difference was statistically significant (p < 0.01, t-test). The standard error and coefficient of variation of MetaVue were 1.07 and 0.195; and for ImageJ were 0.32 and 0.087. The authors conclude that their new approach demonstrates improved convenience, time efficiency, accuracy, and less operator error or bias.

The correlation between calcium intensity and histopathological changes in brachial plexus nerve injuries.

BACKGROUND: After nerve injury, an influx of calcium exceeds the homeostatic capacity, which damages peripheral nerves. Previous studies identified that following nerve crush, function improves as calcium levels normalize. METHODS: Electrophysiological analysis was performed to measure the compound muscle action potential of 15 patients' damaged nerves. These samples were evaluated for calcium level and also stained with a Luxol fast blue and neurofilament antibodies to evaluate the myelin sheath and neurofilaments of the nerves. Based on the Sunderland scale, we identified three exclusive types of peripheral nerve injury groups. RESULTS: There was a correlation between histopathological damage and calcium levels of 0.81 (p < 0.005). The average relative fluorescence units (RFUs) was 235.28 ± 19, which corresponds to 5.3 × 10⁻7 M of calcium, five times the normal value. CONCLUSION: Our study shows promising clinical implications via faster pathology results by the RFU technique. This approach of calcium staining, though still in clinical trials, offers significant clinical application, allowing physicians to get the clinically diagnostic nerve injury degree faster and will also facilitate better strategies for further treatment or future surgeries.

The effect of calcium modulating agents on peripheral nerve recovery after crush.

After a nerve injury, calcium concentration in the intra-nerve fiber drastically increases. The purpose of our study was to test an implantable micro-osmotic pump to deliver medications to accelerate calcium absorption, thereby greatly improving nerve regeneration. Twenty-four SD rats were divided into four groups of six each: (1) Sham control: crush injury to sciatic nerve only; (2) Crush injury with a Nifedipine pump; (3) Crush injury with a Calcitonin pump; (4) Crush injury with a Saline pump. Each rat's right sciatic nerve was crushed. The micro-osmotic pump was implanted in the neck, and the dripping tube was routed to the injured nerve. After four weeks of survival time, compound muscle action potential (CMAP), tetanic muscle force (TMF), myelinated nerve fiber area (NFA), nerve calcium concentration (NCC), and calcified spots (CS) were evaluated. The calcium absorption rate (CAR) was also determined. The order from highest to lowest recovery rate was Nifedipine>Calcitonin>Sham control>Saline. Differences among the groups were statistically significant (P<0.001, ANOVA test), and the difference between Nifedipine/Calcitonin and Saline/Sham control were all statistically significant (P<0.001, t-test). The correlation rate of NCC with CMAP/TMF and with NFA/CS and CAR were calculated to be 0.99 (all P<0.001, Pearson's Correlation). We conclude from this study that nerve regeneration strongly correlated with calcium absorption; our new data has shown greatly improved nerve functional recovery, and this can potentially be translated into clinical applications.

The correlation between calcium absorption and electrophysiological recovery in crushed rat peripheral nerves.

The correlation between calcium ion (Ca2+) concentration and electrophysiological recovery in crushed peripheral nerves has not been studied. Observing and quantifying the Ca2+ intensity in live normal and crushed peripheral nerves was performed using a novel microfine tearing technique and Calcium Green-1 Acetoxymethyl ester stain, a fluorescent Ca2+ indicator. Ca2+ was shown to be homogeneously distributed in the myelinated sheaths. After a crush injury, there was significant stasis in the injured zone and the portion distal to the injury. The Ca2+ has been almost completely absorbed after 24 weeks in the injured nerve to be similar to the controls. The process of the calcium absorption was correlated with the Compound Muscle Action Potential recovery process of the injured nerves. This correlation was statistically significant (r = -0.81, P < 0.05). The better understanding of this process will help us to improve nerve regeneration after peripheral nerve injury.

Qualitative effect on mRNAs of injury-associated proteins by cell phone like radiation in rat facial nerves.

Rats were exposed to cell phone radiation for 6 hours per day for 18 weeks. The buccal and mandibular branches of the facial nerve were evaluated for this study. The mRNA levels of four proteins that are usually up regulated when an injury has occurred were investigated; included were Calcium ATP-ase, Endothelin, Neural Cell Adhesion Molecule, and Neural Growth Factor. These isolated mRNAs were subjected to RT-PCR and all four were up regulated. The mandibular nerve showed a higher and broader level of up regulation than the buccal nerve. All four mRNA up regulations for the mandibular nerve and two for the buccal nerve were also statistically significant. These specific injury-related findings were mild. As the use of these cell phones continues, there most likely will be permanent damage to these tissues over the years and the likelihood of tumors, cancers, and system failures will potentially increase.

Upregulation of specific mRNA levels in rat brain after cell phone exposure.

Adult Sprague-Dawley rats were exposed to regular cell phones for 6 h per day for 126 days (18 weeks). RT-PCR was used to investigate the changes in levels of mRNA synthesis of several injury-associated proteins. Calcium ATPase, Neural Cell Adhesion Molecule, Neural Growth Factor, and Vascular Endothelial Growth Factor were evaluated. The results showed statistically significant mRNA up-regulation of these proteins in the brains of rats exposed to cell phone radiation. These results indicate that relative chronic exposure to cell phone microwave radiation may result in cumulative injuries that could eventually lead to clinically significant neurological damage.

Effects of cellular phone emissions on sperm motility in rats.

OBJECTIVE: To evaluate the effects of cellular phone emissions on rat sperm cells. DESIGN: Classic experimental. SETTING: Animal research laboratory. SUBJECTS: Sixteen 3-month-old male Sprague-Dawley rats, weighing 250-300 g. INTERVENTION(S): Rats in the experimental group were exposed to two 3-hour periods of daily cellular phone emissions for 18 weeks; sperm samples were then collected for evaluation. MAIN OUTCOME MEASURE(S): Evaluation of sperm motility, sperm cell morphology, total sperm cell number, and mRNA levels for two cell surface adhesion proteins. RESULT(S): Rats exposed to 6 hours of daily cellular phone emissions for 18 weeks exhibited a significantly higher incidence of sperm cell death than control group rats through chi-squared analysis. In addition, abnormal clumping of sperm cells was present in rats exposed to cellular phone emissions and was not present in control group rats. CONCLUSION(S): These results suggest that carrying cell phones near reproductive organs could negatively affect male fertility.

Real-time reverse transcriptase polymerase chain reaction: an improvement in detecting mRNA levels in mouse cranial tissue.

BACKGROUND: Quantitation of messenger RNA levels has traditionally been carried out by Northern blot analysis. While this is regarded as the standard method, it is time-consuming and requires large quantities of RNA. Reverse-transcriptase polymerase chain reaction is a semiquantitative method that has been used as a more rapid and sensitive alternative to Northern blotting. Real-time reverse-transcriptase polymerase chain reaction is a quantitative technique that is gaining widespread acceptance as a rapid and reliable way of quantifying mRNA. Since both techniques are currently being used to evaluate gene expression in the murine cranial suture model, the present study was performed to compare the sensitivity and variability of real-time to conventional reverse-transcriptase polymerase chain reaction in this model. METHODS: Mouse brain RNA was isolated and amplified using real-time and conventional methods. For the real-time method, a serial 10-fold dilution of RNA, ranging from 1 fg to 100 ng, was performed. For the conventional method, the minimum amount of RNA needed for consistent polymerase chain reaction amplification was determined. Transforming growth factor beta-1 and beta-actin RNA transcripts were measured using both techniques. RESULTS: One femtogram of RNA could be detected by the real-time method, although 10 fg were required to reliably detect differences; 500 ng of RNA was required for consistent polymerase chain reaction amplification using the conventional method. The variability of real-time reverse-transcriptase polymerase chain reaction when expressed as a coefficient of variation (SD as a percentage of the mean) ranged from 0.23 to 2.6 percent for all genes tested, as compared with 9 to 70 percent for conventional reverse-transcriptase polymerase chain reaction. CONCLUSIONS: Real-time reverse-transcriptase polymerase chain reaction was used successfully to detect mRNA from different mouse genes. The real-time method is much more sensitive in detecting small amounts of mRNA than both Northern blot analysis and conventional polymerase chain reaction. The variability of the real-time method is more than 10-fold lower compared with the conventional method performed in the authors' laboratory for all genes tested.

Detection of apoptosis in fusing versus nonfusing mouse cranial sutures.

Apoptosis may be involved in maintenance of suture patency. In mice, the posterior frontal suture fuses by postnatal day 45, whereas all remaining cranial sutures remain patent. There are no published reports documenting differences in apoptosis between fusing and nonfusing mouse cranial sutures beyond postnatal day 6 either in vivo or in vitro. In the current study, we hypothesized that apoptosis is required for maintenance of suture patency. We predicted that after normal suture fusion in the mouse, the posterior frontal suture should have fewer apoptotic cells than the sagittal suture. We also hypothesized that all of the sutures should look similar with respect to the number and arrangement of apoptotic cells before suture fusion. The posterior frontal and sagittal sutures were studied on postnatal days 25 and 45. The fragmentation of DNA or terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay assay, as well as the presence of BCL-10, a specific apoptotic protein, were localized to the leading edge of the sagittal suture calvaria of postnatal day 45 mice. These apoptotic markers were not visualized within the fused posterior frontal suture of postnatal day 45 mice. Posterior frontal or sagittal suture mesenchyme of postnatal day 25 mice showed similar amounts of apoptotic cells. These data indicate that apoptotic cells are present in the patent sagittal suture beyond the period of posterior frontal suture fusion in the mouse. We conclude that apoptosis is an integral component to maintain suture patency in the mouse calvaria.

TGF-beta1, FGF-2, and receptor mRNA expression in suture mesenchyme and dura versus underlying brain in fusing and nonfusing mouse cranial sutures.

Recent studies have supported a functional role for the transforming growth factor beta-1 (TGF-beta1) and fibro-blast growth factor 2 (FGF-2) signaling cascades in the process of mouse cranial suture fusion. TGF-beta1 and FGF-2 protein expression have been shown to be elevated in the fusing posterior frontal suture versus the nonfusing sagittal suture. The authors evaluated simultaneous mRNA expression of TGF-beta1 and its R1 receptor and FGF-2 and its R2 receptor during mouse cranial suture fusion. They evaluated the suture mesenchyme-dura complex separately from the underlying brain to determine whether there is tissue-specific biologic activity (i.e., brain versus suture mesenchyme-dura) for each cytokine and receptor. Data were collected from 150 male CD-1 mice studied over five time periods from postnatal days 22 to 45. They utilized reverse-transcriptase polymerase chain reaction as a means to detect TGF-beta1, TGF-beta receptor 1 (TGF-betaR1), FGF-2, and FGF receptor 2 (FGFR2) mRNA expression in mouse cranial tissues, beginning with the period of initiation of posterior frontal cranial suture fusion (postnatal day 22) and extending through completion of posterior frontal suture fusion (postnatal day 45). Expression of FGF-2 was significantly greater in posterior frontal suture mesenchyme and dura compared with sagittal suture mesenchyme and dura during the period of initiation of posterior frontal suture fusion, localizing this cytokine's expression to posterior frontal suture mesenchyme and dura during the process of cranial suture fusion. TGF-beta1 and FGFR2 mRNA expression was found to be up-regulated in posterior frontal suture mesenchyme and dura relative to the underlying brain tissue throughout the study period, whereas TGF-betaR1 and FGF-2 mRNA expression was significantly elevated relative to the underlying brain only at time points corresponding to the initiation of posterior frontal suture fusion (between postnatal days 22 and 31). These results indicate that there is tissue-specific mRNA expression of TGF-beta1, FGF-2, and their receptors between suture mesenchyme and dura and the underlying brain, which correlates with the period of posterior frontal suture fusion in the mouse model. Differences in gene expression between suture mesenchyme and dura relative to the underlying brain may be an important regulator of cranial suture biology. Understanding these differences may eventually help to identify possible targets and time windows by which to most effectively modulate cranial suture fusion.