Michael Agresti

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-β1) and fibro-blast growth factor 2 (FGF-2) signaling cascades in the process of mouse cranial suture fusion. TGF-β1 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-β1 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-β1, TGF-β receptor 1 (TGF-βR1), 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-β1 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-βR1 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-β1, 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.

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.

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.

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.

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 rats 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, Pearsons 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.

Qualitative Effect on mRNAs of Injury-Associated Proteins by Cell Phone Like Radiation in Rat Facial Nerves

Rats were exposed to cellphone 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.

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.

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. Muscle Nerve 51: 229–234, 2015

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.

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 &bgr;-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.