Acute intermittent hypoxia enhances regeneration of surgically repaired peripheral nerves in a manner akin to electrical stimulation.

The intrinsic repair response of injured peripheral neurons is enhanced by brief electrical stimulation (ES) at time of surgical repair, resulting in improved regeneration in rodents and humans. However, ES is invasive. Acute intermittent hypoxia (AIH) - breathing alternate cycles of regular air and air with ~50% normal oxygen levels (11% O2), considered mild hypoxia, is an emerging, promising non-invasive therapy that promotes motor function in spinal cord injured rats and humans. AIH can increase neural activity and under moderately severe hypoxic conditions improves repair of peripherally crushed nerves in mice. Thus, we posited an AIH paradigm similar to that used clinically for spinal cord injury, will improve surgically repaired peripheral nerves akin to ES, including an impact on regeneration-associated gene (RAG) expression-a predictor of growth states. Alterations in early RAG expression were examined in adult male Lewis rats that underwent tibial nerve coaptation repair with either 2 days AIH or normoxia control treatment begun on day 2 post-repair, or 1 h ES treatment (20 Hz) at time of repair. Three days post-repair, AIH or ES treatments effected significant and parallel elevated RAG expression relative to normoxia control at the level of injured sensory and motor neuron cell bodies and proximal axon front. These parallel impacts on RAG expression were coupled with significant improvements in later indices of regeneration, namely enhanced myelination and increased numbers of newly myelinated fibers detected 20 mm distal to the tibial nerve repair site or sensory and motor neurons retrogradely labeled 28 mm distal to the repair site, both at 25 days post nerve repair; and improved return of toe spread function 5-10 weeks post-repair. Collectively, AIH mirrors many beneficial effects of ES on peripheral nerve repair outcomes. This highlights its potential for clinical translation as a non-invasive means to effect improved regeneration of injured peripheral nerves.

Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins: comparison with pentosidine.

Methylglyoxal (MG), an alpha-dicarbonyl compound, can be produced in vivo by several metabolic pathways and the Maillard reaction. It reacts rapidly with proteins to form advanced glycation end products or AGEs. We previously isolated and characterized a blue fluorescent product of the reaction between MG and arginine, which we named argpyrimidine. We found that argpyrimidine was stable to acid hydrolysis, which allowed us to hydrolyze tissue proteins with 6 N HCl and quantify argpyrimidine by high-performance liquid chromatography. Here we report argpyrimidine concentrations in human lens and serum proteins as determined by HPLC. We have also measured pentosidine, a fluorescent AGE derived from pentose sugars, and compared the concentrations of pentosidine and argpyrimidine. We found two- to threefold higher argpyrimidine concentrations in diabetic serum proteins than in nondiabetic controls (9.3 +/- 6.7 vs 4.4 +/- 3.4 pmol/mg). We found a significant correlation (P = 0.0001) between serum protein argpyrimidine and glycosylated hemoglobin. Argpyrimidine concentrations were approximately seven times greater in brunescent cataractous lenses than in aged noncataractous lenses. Pentosidine concentrations in serum and lens proteins were much lower than argpyrimidine concentrations; in general, argpyrimidine levels were 10--25 times higher than pentosidine. Results from our study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein-arginine modification by MG in aging and diabetes.

Cellular localization of intestinal calcium-binding protein in pig duodenum.

The gamma-globulin fraction of a rabbit antiserum against porcine intestinal calcium-binding protein (CaBP) was used in an immunoperoxidase method to study CaBP localization in porcine duodenal tissue. Specific immunostaining, indicative of the presence of CaBP, was noted within the cytoplasm of the duodenal epithelial cells. No CaBP was detected in goblet cells or in the subepithelial layers. When the specific antibody was replaced by either nonimmune rabbit gamma-globulin or when the specific antibody was preadsorbed with excess CaBP, no positive immunostaining was seen. Our studies lend support to the hypothesis that CaBP may function in intracellular calcium transport.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. I. Identification of intestinal calcium-binding protein in blood and response to a low calcium diet.

We have developed a radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) and have used it to detect CaBP in pig plasma. Plasma CaBP is identical to intestinal CaBP on the basis of immunological activity, molecular size, and molecular charge properties. The plasma CaBP concentration was greater in the portal blood than in mixed venous blood, suggesting that blood CaBP originates in the gut. Two of four 15-week-old littermate pigs were placed on a low calcium diet (0.15% calcium, 0.65% phosphorus) and two on a control diet (0.65% calcium, 0.65% phosphorus). After 2 weeks, the entire small intestine was removed and divided into nine 1.8-m segments. CaBP was assayed in both plasma and intestinal mucosa. When the two pigs on a low calcium diet were compared with two control pigs, there was a general increase in immunoreactive CaBP in both plasma and intestinal mucosa. However, there was no increment in immunoreactive CaBP in the first 1.8-m segment of small intestine. Seventy-one percent of the increment in CaBP occurred distal to the first two segments. The largest fractional low calcium diet effect occurred in the ileum. The mean CaBP concentration for the total small intestine increased by a factor of 1.9. The plasma CaBP concentration increased by a factor of 2.6. In these pigs, plasma CaBP was a more reliable indicator of change in CaBP status than was the measurement in the proximal gut segment which contained the duodenum. The assay of CaBP in blood is convenient and may obviate the sampling errors inherent in intestinal biopsy.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. II. The distribution of intestinal CaBP in pig tissues.

Using a specific radioimmunoassay for porcine intestinal calcium-binding protein (CaBP), we have measured the concentration of CaBP in the various tissues and organs of normal pigs. Intestinal CaBP was present in highest concentration in the upper small intestine, with lower concentrations in the distal small intestine. Intestinal CaBP was also found, in lower concentrations, in kidney, liver, thyroid, pancreas, and blood. In all other tissues, including parathyroid, bone, skeletal muscle, and brain, CaBP immunoreactivity was undetectable or less than in blood. The elution profile of calcium-binding activity and immunoreactivity from gel filtration analysis of kidney and parathyroid extracts suggest that the calcium-binding protein in the parathyroid gland, and the major calcium-binding protein(s) in the kidney, are chemically and immunochemically different from intestinal CaBP.

Apathetic thyrotoxicosis.

Apathetic thyrotoxicosis is an atypical though not rare manifestation of hyperthyroidism. The cardinal features are apathy and depression, as opposed to hyperkinesis and mental alertness in the usual thyrotoxic patient, and are unassociated with the usual signs and symptoms of hyperthyroidism, making the diagnosis difficult. We report three cases of apathetic thyrotoxicosis seen during one year.