Transient activation of hedgehog pathway rescued irradiation-induced hyposalivation by preserving salivary stem/progenitor cells and parasympathetic innervation.

PURPOSE: To examine the effects and mechanisms of transient activation of the Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer. EXPERIMENTAL DESIGN: Mouse salivary glands and cultured human salivary epithelial cells were irradiated by a single 15-Gy dose. The Hedgehog pathway was transiently activated in mouse salivary glands, by briefly overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), parasympathetic innervation, and expression of related genes were examined by flow cytometry, salisphere assay, immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA. RESULTS: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the Bmi1 and Chrm1-HB-EGF pathways. The preservation of parasympathetic innervation was associated with the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation. CONCLUSIONS: These findings suggest that transient activation of the Hedgehog pathway has the potential to restore salivary gland function after irradiation-induced dysfunction.

Parasympathetic stimulation improves epithelial organ regeneration.

Parasympathetic nerves are a vital component of the progenitor cell niche during development, maintaining a pool of progenitors for organogenesis. Injured adult organs do not regenerate after parasympathectomy, and there are few treatments to improve organ regeneration, particularly after damage by therapeutic irradiation. Here we show that restoring parasympathetic function with the neurotrophic factor neurturin increases epithelial organ regeneration after damage. We use mouse salivary gland explant culture containing fluorescently labelled progenitors, and injure the tissue with irradiation. The progenitors survive, parasympathetic function is diminished and epithelial apoptosis reduces the expression of neurturin, which increases neuronal apoptosis. Treatment with neurturin reduces neuronal apoptosis, restores parasympathetic function and increases epithelial regeneration. Furthermore, adult human salivary glands damaged by irradiation also have reduced parasympathetic innervation. We propose that neurturin will protect the parasympathetic nerves from damage and improve organ regeneration. This concept may be applicable for other organs where parasympathetic innervation influences their function.

Bilateral loss of light reflex and accommodation following 360° peripheral retinal laser therapy.

We report a case of bilateral loss of pupillary light reflex and accommodation following 360° peripheral retinal laser therapy. A 24 years old male underwent prophylactic laser barrage for peripheral retinal lattice degenerations. Soon after the procedure, he developed bilateral loss of pupillary light reflex and accommodation. The patient faced difficulty while doing near work. On instillation of 0.125% pilocarpine, both pupils demonstrated the phenomenon of denervation supersensitivity. Damage to the short ciliary nerves was the most likely mechanism responsible for this adverse outcome.

Spectral sensitivity of the photointrinsic iris in the red-eared slider turtle (Trachemys scripta elegans).

Our goal in this study was to examine the red-eared slider turtle for a photomechanical response (PMR) and define its spectral sensitivity. Pupils of enucleated eyes constricted to light by ∼11%, which was one-third the response measured in alert behaving turtles at ∼33%. Rates of constriction in enucleated eyes that were measured by time constants (1.44-3.70 min) were similar to those measured in turtles at 1.97 min. Dilation recovery rates during dark adaptation for enucleated eyes were predicted using line equations and computed times for reaching maximum sizes between 26 and 44 min. Times were comparable to the measures in turtles where maximum pupil size occurred within 40 min and possessed a time constant of 12.78 min. Hill equations were used to derive irradiance threshold values from enucleated hemisected eyes and then plot a spectral sensitivity curve. The analysis of the slopes and maximum responses revealed contribution from at least two different photopigments, one with a peak at 410 nm and another with a peak at 480 nm. Fits by template equations suggest that contractions are triggered by multiple photopigments in the iris including an opsin-based visual pigment and some other novel photopigment, or a cryptochrome with an absorbance spectrum significantly different from that used in our model. In addition to being regulated by retinal feedback via parasympathetic nervous pathways, the results support that the iris musculature is photointrinsically responsive. In the turtle, the control of its direct pupillary light response (dPLR) includes photoreceptive mechanisms occurring both in its iris and in its retina.

Photoperiod alters autonomic regulation of the heart.

Outside of the tropics, environmental conditions fluctuate in a generally predictable manner across the year. Many small mammals have evolved mechanisms, such as seasonal breeding and annual adjustments in physiology, morphology, and behavior, that promote winter survival when food is scarce and thermoregulation is challenging. Photoperiod (day length) is a cue used by many seasonal breeders to predict seasonal changes in environmental conditions. One system that is uniquely situated to mediate photoperiod-induced alterations in physiology is the autonomic nervous system (ANS). The 2 branches of the ANS are key regulators of immune responses, thermoregulation, and energy balance, functions that undergo marked shifts in baseline and reactivity following acclimation to short day lengths. Although previous studies have investigated the effects of photoperiod on ANS endpoints, this study examined the direct effects of photoperiod on integrated ANS function. To test the hypothesis that short day lengths increase parasympathetic and sympathetic tones, we maintained adult male Siberian hamsters (Phodopus sungorus) to either long or short photoperiods and then analyzed electrocardiogram recordings. Short day lengths increased both parasympathetic tone, as measured by respiratory sinus arrhythmia, and sympathetic control of the heart, measured with autonomic blockade. Additionally, short day lengths enhanced the withdrawal of parasympathetic control and the increase of sympathetic tone in response to acute restraint stress. Finally, these effects were discovered to be independent of circulating androgens. These data indicate that the ANS of Siberian hamsters undergoes profound changes following prolonged exposure to short winter-like day lengths.

[Effect of radiotherapy on heart rate stability in cancer patients].

Heart rate variability at rest and in active orthostatic test (AOT), before and after radiotherapy (STD 3Gy; TTD 30Gy), was studied in 4 left-breast cancer patients and 8 cases of esophageal (middle third) cancer. AOT involved by decreased baroreflective activity (7) and parasympathetic reactivity. Sympathoadrenal fraction was relatively prominent in 6 patients against the background of average declined heart rate (3). Prior to radiotherapy, average heart rate was low in all patients, and continued to fall in 10 patients after treatment. While tumor process inhibited functional status and systemic reactions of adaptation, they were still further depressed by radiotherapy resulting eventually in failure of such response.

Parasympathetic control of the pupillary light response in the red-eared slider turtle (Pseudemys scripta elegans).

OBJECTIVE: We investigated effects of both vecuronium bromide, a nicotinic cholinergic antagonist, and atropine, a muscarinic cholinergic antagonist, on the pupil of the turtle to determine whether responses to light are controlled by parasympathetic innervations acting on the iris. ANIMAL STUDIED: Three red-eared slider turtles, Pseudemys scripta elegans. PROCEDURE: Turtles were secured to immobilize their head movements and then inserted into a light-integrating sphere. Both pupils were monitored through small apertures by digital video cameras. Pupil diameters were measured manually with a digital caliper. During each trial, drugs (0.4%) were topically applied, four times at 15 min intervals, to the corneas of each eye. One eye was randomly selected for treatment of the drug while the other, treated with saline (0.9% NaCl), was used as control. Pupil sizes under adaptation to light were tracked after drug or saline applications. RESULTS: Mean pupillary diameters of eyes treated with vercuronium bromide increased by 28%, reaching peak size in 90 min. Onset of response occurred 20 min after drug application and then increased at a rate having a time constant of 26 min. Recovery began at 120 min after initial application. Atropine had no effect on pupil size. No systemic side effects by drugs were observed in turtles. CONCLUSIONS: Although atropine does not cause mydriasis, vecuronium bromide does. These results suggest that the parasympathetic system in turtles acts through acetylcholine onto nicotinic receptors to stimulate pupillary light constriction.

Heart rate variability in workers exposed to medium-frequency electromagnetic fields.

This study was undertaken to evaluate the neurovegetative regulation of the heart in workers occupationally exposed to medium frequency (MF) electromagnetic (EM) fields. The subjects were 71 workers of MF broadcast stations, aged 20-68 (mean 47.1) with the duration of work under exposure ranging from 2-40 years and 22 workers of radio link stations, aged 21-65 (mean 46.9) who were not exposed to MF EM fields. The distribution of age and work tenure in both groups did not differ significantly. Heart rate variability (HRV) was analysed basing on 512 normal heart evolutions registered in resting, from the body surface, using the Medea-HRV system. The analysis concerned time-domain and frequency-domain parameters of HRV using fast fourier transformation. Power spectrum in the low (0.05-0.15 Hz) and high (0.15-0.35 Hz) frequency bands (LF and HF, respectively) was determined. Statistically insignificant differences found between exposed and non-exposed groups were found either in time- or in frequency-domain parameters of HRV. No correlation between the power spectrum and the subjects age was noted. Such a relationship, however, could be observed in the control group. In the study group a statistically significant negative correlation was found between the maximum intensity of EM fields and HF power spectrum. Thus it was concluded that occupational exposure to EM fields brings about impairments in the neurovegetative regulation of the cardiovascular function.

[The dependence of the parasympathetic reaction of the mesencephalic section of the brain stem to age-, sex- and pigment-reagent-related factors]. / Zavisimost' parasimpaticheskoi reaktsii mezentsefal'nogo otdela stvola ot vozrastnogo, polovogo i pigment-reagentnogo faktorov.

Photoelectron pupillograph measurements of pupillary responses to light were made in 198 healthy individuals. Photoreaction of the pupils was declining with age contrary to the reciprocity rate which was stable (3.01-3.56). It indicates on the relative stability of parasympathetic-sympathetic relations. Young males with hyperpigmented irises had hyperactive parasympathetic reaction which sharply decreased to normal values at the age of 26-30 years. Estimated standard values of time, strength and speed of pupillomotor reaction can be used for comparative evaluation of autonomic neuromotor and brain structures involvement in various neurological diseases. These values may serve as initial point for devising medical algorithms and computer programs.

[The effect of ionizing radiation on the nervous mechanisms regulating cardiac activity]. / Vplyv ionizuiuchoï radiatsiï na nervovi mekhanizmy rehuliatsiï diial'nist' sertsia.

Functional state of cardiac nervous links and pattern of cardiogenic reflectory responses to radiation (4 Gy) were studied in acute experiments on anesthetized rats at various periods after single radiation. The control level of nervous activity significantly varies in different terms after radiation. Intensification of parasympathetic effects and domination of depressor reactions are typical of the initial period (the 1st day). A decrease in the sympathetic tone and weakening of adrenergic influences are observed in all terms after radiation (from the 1st till the 14th day). Interrelationships between sympathetic and parasympathetic links of heart regulation as well as cardiogenic reflectory responses are not restored to the control level in remote terms after radiation (the 14th-20th days). So, single radiation of animals in a dose of 4 Gy has induced a disorder in the nervous heart regulation, which may be a cause of different pathological states.

Effect of amplitude-modulated radio frequency radiation on cholinergic system of developing rats.

We examined the effect of long-term exposure to radio frequency radiation 147 MHz and its sub-harmonics 73.5 and 36.75 MHz amplitude modulated at 16 and 76 Hz (30-35 days, 3 h per day) on cholinergic systems in developing rat brain. A significant decrease in acetylcholine esterase activity was found in exposed rats as compared to the control. Decrease in acetylcholine esterase (AChE) activity was independent of carrier wave frequencies. A short-term exposure did not have any significant effect on AChE activity.

Bilateral irradiation of head and neck induces an enhanced expression of substance P in the parasympathetic innervation of the submandibular gland.

Substance P and calcitonin gene-related peptide (CGRP) are present in nerve fibers innervating the submandibular gland. Radiotherapy of tumors in the head and neck region usually embraces the salivary glands in the irradiated field and consequently a dramatic decrease in salivary function is seen. In this study, the submandibular glands and ganglia of rats subjected to fractionated irradiation were examined by use of immunohistochemical techniques for demonstration of substance P and CGRP. Irradiation was given on five consecutive days (daily doses of 6-9 Gray) with unilateral or bilateral irradiation techniques. Specimens of control and experimental animals were processed in parallel. A marked increase in the expression of substance P in the ganglionic cells--presumably parasympathetic--and in the number of fibers showing substance P-like immunoreactivity in association with acini and small ducts was seen in response to bilateral irradiation. (Surprisingly, unilateral irradiation of the parotid area had no effect on peptide distribution in the irradiated gland and ganglion). No changes in the pattern of CGRP immunoreactivity occurred. In the trigeminal ganglion, which supplies the submandibular gland with the majority of the sensory substance P- and CGRP-containing nerve fibers, no changes in the expression of substance P or CGRP immunoreactivity were seen. The results suggest that bilateral irradiation leads to an increase in the synthesis of substance P-like substance in the parasympathetic ganglionic cells supplying the submandibular gland with secretory nerves, and can thus be an additional factor in explaining the altered secretory capacity of salivary glands.

[The characteristics of the radiation lesion in exposure of the anterior abdomen in dogs and the effect of modifying factors]. / Osobennosti luchevogo porazheniia pri vozdeistvii na perednii otdel zhivota sobak i vliianie nekotorykh modifitsiruiushchikh faktorov.

A comparative study was made of the effect of gamma/neutron and X-radiation on the forepart of dog abdomen. The RBE coefficient was found to depend upon body mass and to range from 1.3 to 1.5. The exclusion of the sympathetic nervous system before irradiation was shown to aggravate the clinical condition and to increase the death rate. On the contrary, the exclusion of the parasympathetic nervous system before irradiation and gastric lavage after irradiation improved the clinical picture of radiation sickness and increased the radioresistance of dogs.

Parasympathetic denervation of the ciliary muscle following retinal photocoagulation.

Cynomolgus monkeys underwent unilateral PRP with xenon arc or argon or krypton laser light, employing burn intensity, size, spacing, and topography analogous to standard clinical (eg, Diabetic Retinopathy Study) treatment. Shortly thereafter, accommodative responsiveness to topical eserine and electrical stimulation of the EWN was diminished, accommodative responsiveness to systemic pilocarpine was enhanced, and the number of muscarinic receptors in the ciliary muscle was reduced in the PRP-treated eyes compared to the contralateral controls. In most instances, these parameters returned to normal over 6 to 12 weeks and the abnormalities could be induced again by another round of PRP. However, in some PRP-treated eyes, accommodative responsiveness to EWN stimulation and topical eserine remained subnormal permanently (greater than 1 year). Light and electron microscopy of the ciliary muscle and choroid confirmed the early interruption and degeneration and the subsequent regeneration of the intraocular parasympathetic nerves following PRP. These findings are similar to those seen after surgical removal of the ciliary ganglion and posterior ciliary nerves, and indicate that PRP produces an intraocular parasympathetic denervation of the ciliary muscle. This phenomenon may explain the loss of voluntary accommodation which can follow PRP in prepresbyopic humans. Three cynomolgus monkeys underwent nasal and temporal HRMP in one eye with the argon laser. One to four weeks later, accommodative responses to IM pilocarpine, topical eserine, and electric stimulation of the EWN did not differ markedly in the treated and control eyes. Five weeks after HRMP, posterior PRP was performed in the same eye, sparing the previously treated areas. One to four weeks later, accommodative responses in the PRP-treated eyes were clearly subsensitive to central electrical stimulation, but supersensitive to IM pilocarpine, compared to the contralateral controls. These findings indicate that extensive parasympathetic denervation of the ciliary muscle occurs following PRP but not following HRMP. Consequently, we infer that parasympathetic motor nerve fibers to the ciliary muscle do not travel preferentially with the LPCN, but rather travel primarily if not exclusively with the more numerous SPCN. If the monkey and the human are comparable, sparing the horizontal retinal meridians during clinical PRP so as not to disturb the LPCN will probably not help to preserve ciliary muscle function and accommodation.

Low-level microwave irradiation and central cholinergic systems.

Our previous research showed that 45 min of exposure to low-level, pulsed microwaves (2450-MHz, 2-microseconds pulses, 500 pps, whole-body average specific absorption rate 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. The effects of microwaves on central cholinergic systems were further investigated in this study. Increases in choline uptake activity in the frontal cortex, hippocampus, and hypothalamus were observed after 20 min of acute microwave exposure, and tolerance to the effect of microwaves developed in the hypothalamus, but not in the frontal cortex and hippocampus, of rats subjected to ten daily 20-min exposure sessions. Furthermore, the effects of acute microwave irradiation on central choline uptake could be blocked by pretreating the animals before exposure with the narcotic antagonist naltrexone. In another series of experiments, rats were exposed to microwaves in ten daily sessions of either 20 or 45 min, and muscarinic cholinergic receptors in different regions of the brain were studied by 3H-QNB binding assay. Decreases in concentration of receptors occurred in the frontal cortex and hippocampus of rats subjected to ten 20-min microwave exposure sessions, whereas increase in receptor concentration occurred in the hippocampus of animals exposed to ten 45-min sessions. This study also investigated the effects of microwave exposure on learning in the radial-arm maze. Rats were trained in the maze to obtain food reinforcements immediately after 20 or 45 min of microwave exposure.(ABSTRACT TRUNCATED AT 250 WORDS)