Light Microscopic Autoradiography Research Articles

Biodistribution, cellular localization, and in vivo tolerability of 35S-labeled antiinflammatory dendritic polyglycerol sulfate amine

Antiinflammatory dendritic polyglycerol sulfate (dPGS) holds great potential in the treatment and imaging of inflammatory processes. Here, we studied its biokinetic behavior, biodistribution, target cells, and in vivo toxicology. Following intravenous or subcutaneous application of 35sulfur-labeled dPGS amine with a molecular weight of 10.05 kDa and a hydrodynamic diameter of 5.7 ± 1.5 nm to mice, tissues were collected at specific time points (2, 15 min; 1, 24 h; 5, 21 days) and analyzed by liquid scintillation counting, autoradiography, radioluminography, and light microscopic autoradiography. The blood half-life of dPGS amine was 12 days. The major route of elimination was via the bile and feces. Elimination via the kidney and urine was only initially observed after i.v., but not after s.c. injection. Regardless of the administration mode, liver and spleen were late target organs where dPGS amine accumulated in phagocytic cells. Despite bioaccumulation, toxicological histopathology failed to identify any adverse effects at any time and in any tissues examined suggesting a high in vivo biocompatibility and encouraging future investigation for biomedical applications.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques.

The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

The direct renin inhibitor aliskiren localizes and persists in rat kidneys

The aims of this study were to 1) determine whether renal localization of aliskiren and its antihypertensive and renoprotective effects persist after administration of the drug is stopped and 2) define the renal localization of aliskiren by light microscopy autoradiography. Hypertensive double transgenic rats (dTGR) overexpressing genes for human renin and angiotensinogen were treated with aliskiren (3 mg·kg(-1)·day(-1) sc; osmotic minipumps) or enalapril (18 mg/l in drinking water). After a 2-wk treatment, dTGR were assigned to either continued treatment with aliskiren ("continued"), or to cessation of their respective treatment ("stopped") for a 3-wk washout. One week of treatment with aliskiren and enalapril reduced blood pressure and albuminuria vs. baseline. After cessation of either treatment, blood pressure had returned to pretreatment levels and albuminuria remained relatively low for 1 wk, but rose thereafter similarly in both groups. In contrast, renal mRNA for transforming growth factor-β and renal collagen IV was reduced by aliskiren (continued and stopped groups), but not after cessation of enalapril. Similar patterns were found for collagen IV protein expression. Even 3 wk after stopping aliskiren treatment, renal levels of the drug exceeded its IC50, whereas enalaprilat was not detected. To localize aliskiren accumulation, Wistar rats were treated with [(3)H]-aliskiren for 7 days. Autoradiography demonstrated specific labeling in glomeruli, arterioles, and afferent arterioles as well as in the distal nephron. Labeling could still be observed even after 7 days' washout. These results suggest that the renophilic properties of aliskiren are different from enalapril and could have contributed to the renoprotective mechanism of this renin inhibitor.

Tissue and cellular localization of nanoparticles using 35S labeling and light microscopic autoradiography

Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of 35S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG35S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. From the Clinical EditorIn this study, a novel light microscopic autoradiography utilizing 35S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard.

New route for Tizanidine administration: a pharmacokinetics and light microscope autoradiography study

Spasticity could represent a complication of several central nervous system (CNS) disorders. Multiple sclerosis and painful paroxysmal syndromes (e.g. trigeminal neuralgia) are pathologies in which anti-spastic drugs are used to a greater extent . Baclofen, tizanidine (TIZ), benzodiazepines, dantrolene, and, more recently, gabapentin are the pharmacological agents more widely used. Baclofen, TIZ, benzodiazepines, gabapentin, clonidine but not dantrolene are active on CNS. The alpha2 adrenoceptor agonist TIZ is one of the most effective and largely used anti-spastic drugs. Oral treatment is the only route of anti-spastics administration, although it may cause problems of bioavailability and/or compliance in spastic patients with impaired deglutition. This study was designed to assess the possibility of develop a new route of administration of TIZ and to identify its targets in the spinal cord. New Zealand rabbits were treated with oral (OR , n=6), intramuscular (IM, n=6) or intranasal (IN, n=6) TIZ (3.2 mg/kg/day). Plasma concentration was measured by HPLC on samples collected at 0, 30, 60, 90, 120 and 480 min after treatment. Curves of average concentrations of TIZ vs. time were constructed. In plasma, TIZ reached a peak between 45-65 min after administration. C max was in the range of 268.33–1213.64 ng/ml for IM treatment, of 73.95–135.92 ng/ml for IN treatment and of 16.86–857.25 ng/ml for OR treatment. After pharmacokinetic studies, different spinal cord tracts were removed and used for radioligand binding assay and autoradiography. Using [3H]-RX821002 ([3H]-RX) (0.1-14nM), the Kd and Bmax for every spinal cord segment were calculated. The non-specific binding was obtained with 100µM of (-)-epinephrine. [3H]-RX sites were accumulated in the superficial laminae of dorsal horn. Dense [3H]-RX binding in control was seen over the superficial dorsal horn (laminae I-II) and centrally located lamina X. The ventral horn showed moderate levels of binding. [3H]-RX was displaced after TIZ treatment using different administration routes. The heaviest accumulation of silver grains (lowest displacement of TIZ) occurred after OR administration of the compound, the lowest after IM administration. These results suggest that both IM and IN administration of TIZ may represent routes of administration of the drug alternative to the OR one. Considering predictable adverse effects of IM treatment, the IN administration could represent the elective route to administrate this kind of drugs.

Distribution of benzo[a]pyrene in discrete regions of rat brain tissue using light microscopic autoradiography and gamma counting

In order to study the distribution of benzo[a]pyrene (B[a]P) in rat brain, Sprague-Dawley male rats, 21-day-old, weighted 40–50 g were given a single intravenous (iv) injection of 3.7 × 105 Bq kg−1 of 14C-B[a]P. Rats were sacrificed at 1, 6, 12, 24, or 48 h after the administration of radiolabeled B[a]P. Light microscopic autoradiography and gamma counting were used. Gamma counting and light microscopic autoradiography showed that 14C-B[a]P localized in hippocampus at 1 h, in cerebral cortex at 6 h, and corpus striatum at 24 h after treatment. After B[a]P administration, silver grains rose with time reaching a peak at 24 h and sharp decrease at 48 h. The silver grains distribution was higher in neurons than in gliocytes. Thus, B[a]P penetrated the blood-brain barrier and distributed in various regions of rat brain.

Tamoxifen-Induced Adduct Formation and Cell Stress in Human Endometrial Glands

The beneficial effects of tamoxifen in the prevention and treatment of breast cancer are compromised by an increased risk of endometrial polyps, hyperplasia, and cancer. Tamoxifen is metabolized to an array of metabolites with estrogenic effects but also to reactive intermediates that may form protein and DNA adducts. The aim of this study was to investigate cellular [(3)H]tamoxifen adduct formation by light microscopic autoradiography and cell stress by immunohistochemical analysis of glucose-regulating protein 78 (GRP78), nuclear factor kappaB (NF-kappaB), and caspase 3 in human endometrial explants after short-term incubation with tamoxifen. The cellular expression of tamoxifen-metabolizing enzymes in human endometrial biopsy samples was also determined by immunohistochemistry. The results showed selective [(3)H]tamoxifen adduct formation in glandular and surface epithelia after incubation with a nontoxic concentration of [(3)H]tamoxifen (6 nM). There was also a selective expression of the endoplasmic reticulum stress chaperone GRP78 and activated caspase 3 at these sites after incubation with cytotoxic concentrations of tamoxifen (10-100 microM). The cell stress was preferentially observed in samples from women in the proliferative menstrual phase. No treatment-related expression of NF-kappaB was observed. Constitutive expression of the tamoxifen-metabolizing enzymes CYP1B1, CYP2A6, CYP2B6, CYP2C8/9/19, CYP2D6, and SULT2A1 in glandular and surface epithelia was shown, but there was a large interindividual variation. The colocalization of [(3)H]tamoxifen adducts, expression of GRP78, caspase 3, and tamoxifen-metabolizing enzymes in human glandular and surface epithelia suggest a local bioactivation of tamoxifen at these sites and that epithelial cells are early target sites for tamoxifen-induced cell stress.

The influence of exogenous oxytoxin on labelling of secretory epithelium of mouse mammary gland by [3H]leucine, evidenced by autoradiography.

It has been suggested that oxytocin, besides its milk-ejecting activity, is also involved in the hormonal regulation of the mammary gland secretory cells. The available data, however, are conflicting. In this study two independent experiments, separated by a certain time interval show that oxytocin intravenously administered to mice at days 10-14 of lactation diminished the incorporation of [3H]leucine into the mammary gland tissue by 32 and 53 per cent, respectively. The neurohormone was co-injected with the tracer amino acid. The radioactivity taken up by the secretory cells was estimated by light microscopic autoradiography. The autoradiograms were evaluated by visual silver grain counting. Tissue radioactivity was measured by liquid scintillation counting. A milk stasis in the mammary gland induced by depriving the mice of suckling two hours before tracer injection had no influence on the secretory activity of the glandular cells. It is assumed that oxytocin has a direct effect on the milk-producing cells, and that the reduction in measurable radioactivity caused by the neurohormone may be due to an accelerated intracellular passage of labelled milk proteins.

Effects of Amifostine Administration Prior to Irradiation to the Submandibular Gland in Mice: Autoradiographic Study using 3H-leucine

We histochemically investigated the radioprotection of organelle in acinus on the submandibular gland using amifostine. Mice were divided into those without amifostine administration or irradiation (group A), with amifostine administration and without irradiation (group B), without amifostine and with 5 Gy of irradiation (group C), and with amifostine and 5 Gy of irradiation (group D). In groups B and D were given 100 mg/kg of amifostine at 30 minutes prior to irradiation to the area equivalent to the submandibular gland. HE staining revealed that group D showed atrophy of the acinar cells, though the change was milder than that seen in group C. In AZAN-Mallory staining, fibrogenesis were found more often in group C as compared to the other groups. In Light Microscopic Autoradiography findings, the ratio of reduced silver particles in the acinar cells was lower in group C than in the other groups at 30 minutes after RI administration. Microstructure findings showed the findings were very similar to those of group A. In Electron microscopic Autoradiography, the ratio among the secretory granules was lower in group C at 120 minutes after RI administration. Our results indicate that amifostine can alleviate xerostomia caused by late effect from radiation.

Effect of Preliminary Amifostine Administration in Irradiation of Parotid Glands

To investigate the effect of amifostine, we administered amifostine, a radioprotective agent, 30 minutes before exposing the maxillary region of mice, including the parotid gland, to 5 Gy or 10 Gy X-ray irradiation. The survival rate was recorded, and changes in the parotid gland morphology were investigated by examining the hematoxylin and eosin (HE)-stained specimens and light microscope autoradiography (LMARG) images obtained 30 days after irradiation. A survival rate of 100% was not observed in any group administered 200 mg/kg amifostine with or without irradiation. Among the groups irradiated with 10 Gy X-rays, the survival rate was higher and the survival period was longer in the 100 mg/kg amifostine group than in the no amifostine group. The histological findings in the group that received 5 Gy irradiation without amifostine were as follows : auxetic growth of acinar cells, nuclei of all sizes, cells in the mitotic phase, and cells undergoing apoptosis. Further, the treated groups were compared with the no amifostine and no irradiation group (untreated control). LMRG imaging revealed that the number of reduced silver grains per mm2 of acinar cells after 30 min of 3H-leucine administration was higher than that after 120 min in mice treated with 100 mg/kg amifostine with or without 5 Gy irradiation. This observation was similar to that in the untreated control.This finding suggests that although amifostine administration reduces the adverse effects of irradiation on the parotid gland, higher doses of amifostine may be fatal.

Isoproterenol Protects the Demilune Cells of the Sublingual Gland of Mice from Radiation: Study of Autoradiography with 3H-leucine

In order to examine the influence of X-ray irradiation on the demilune cells of the sublingual gland due to the existence of secretory granules, 10 Gy of X-ray irradiation was applied to the sublingual gland of mice at 3 hours after isoproterenol (IPR) administration. To inspect the influence of irradiation at 3 days after the irradiation, tissue images and results of autoradiography performed at 30 and 120 minutes (min) after 3H-leucine administration of the IPR administration (IPR/10Gy) group were compared with those of the non-IPR administration (nonIPR/10Gy) group. In transmission electron microscope images, swelling and pyknosis were observed in the rough endoplasmic reticulum of the nonIPR/10Gy group. The number of reduced silver grains per unit cell area in nonIPR/10Gy at 30 and 120 min after 3H-leucine administration was less and greater than that in the other 3 groups (nonIPR/0Gy, IPR/0Gy, and IPR/10Gy), respectively, from light microscope autoradiography images. At 120 min after 3H-leucine administration, the ratio of the number of reduced silver grains localized in the secretory granules to the total number of reduced silver grains in the demilune cells of the nonIPR/10Gy group was lower than that of the other 3 groups as indicated by electron microscope autoradiography images. Based on these results, it was apparent that the effect of irradiation was less on the demilune cells that discharged secretory granules than those that did not discharge them.

Pudendal motoneurons of the rat located in separated spinal nuclei possess nicotinic acetylcholine receptors having distinct pharmacological profiles

Pudendal motoneurons are located in the ventral horn of the caudal lumbar spinal cord and innervate striated pelvic muscles implicated in sexual and eliminative functions. In rats they are distributed in the dorsomedial (DM) and dorsolateral (DL) nucleus. In male rats, dorsomedial motoneurons innervate the bulbocavernosus, the levator ani and the external anal sphincter, whereas dorsolateral motoneurons control the ischiocavernosus and external urethral sphincter. Using spinal cord slices of young male rats and whole-cell patch-clamp recordings, we investigated the sensitivity of pudendal motoneurons to nicotinic cholinergic agonists. Motoneurons were identified following 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulphonate retrograde labelling. In the presence of atropine, both dorsomedial and dorsolateral motoneurons responded to acetylcholine (ACh) by generating a rapidly activating inward current. By using selective nicotinic antagonists and a nicotinic positive allosteric modulator, we found that nicotinic ACh receptors present in dorsomedial and dorsolateral motoneurons display distinct pharmacological profiles. Whereas the former are of the heteromeric type, the latter are predominantly of the alpha7-containing type. These data were confirmed by light microscopic autoradiography. In young rats, a ligand for heteromeric nicotinic receptors labelled all laminae of the central grey matter, whereas in the ventral part of the central grey, a ligand selective for alpha7-containing nicotinic receptors labelled the DL but not the DM. Dorsolateral and dorsomedial motoneurons innervate two distinct groups of pelvic muscles. A difference in their nicotinic pharmacology may be clinically relevant, as it might allow a selective pharmacological intervention in view of influencing the activity of one or the other set of muscles.

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.

Nuclear and Extranuclear Estrogen Binding Sites in the Rat Forebrain and Autonomic Medullary Areas

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

Species differences in 3-methylsulphonyl-DDE bioactivation by adrenocortical tissue

The CYP11B1-activated adrenocortical toxicant 3-methylsulphonyl-DDE (3-MeSO2-DDE) is proposed as a lead compound for an improved chemotherapy for adrenocortical carcinoma. We compared the binding of 3-MeSO2-[14C]DDE in the adrenal cortex of four rodent species; hamster, guinea pig, mouse and rat, using a precision-cut adrenal slice culture system ex vivo. Localization and quantification of the bound radioactivity were carried out using light microscopy autoradiography and radioluminography. The results revealed major species differences since 3-MeSO2-[14C]DDE was extensively bound to the hamster adrenal tissue while the guinea pig adrenals were devoid of binding. A high binding in mouse adrenal cortex was confirmed while binding in rat adrenal cortex was very weak. The results support previous observations that metabolic activation of 3-MeSO2-DDE is highly species dependent. Since CYP11B1 could be expressed in tissues other than the adrenal cortex, final toxicological characterization should be carried out in a species that can bioactivate this compound.

Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanisms

The lateral septal area is rich in vasopressin V(1A) receptors and is densely innervated by vasopressinergic axons, originating mainly from the bed nucleus of the stria terminalis and the amygdala. Genetic and behavioral studies provide evidence that activation of vasopressin receptors in this area plays a determinant role in promoting social recognition. What could be the neuronal mechanism underlying this effect? Using rat brain slices and whole-cell recordings, we found that lateral septal neurons are under the influence of a basal GABAergic inhibitory input. Vasopressin, acting via V(1A) but not V(1B) receptors, greatly enhanced this input in nearly all neurons. The peptide had no effect on miniature inhibitory postsynaptic currents, indicating that it acted on receptors located in the somatodendritic membrane, rather than on axon terminals, of GABAergic interneurons. Cell-attached recordings showed that vasopressin can cause a direct excitation of a subpopulation of lateral septal neurons by acting via V(1A) but not V(1B) receptors. The presence in the lateral septum of V(1A) but not of V(1B) receptors was confirmed by competition binding studies using light microscopic autoradiography. In conclusion, vasopressin appears to act in the lateral septum in a dual mode: (i) by causing a direct excitation of a subpopulation of neurons, and (ii) by causing an indirect inhibition of virtually all lateral septal neurons. This modulation by vasopressin of the lateral septal circuitry may be part of the neuronal mechanism by which the peptide, acting via V(1A) receptors, promotes social recognition.

The vasopressin‐induced excitation of hypoglossal and facial motoneurons in young rats is mediated by V1a but not V1b receptors, and is independent of intracellular calcium signalling

As a hormone, vasopressin binds to three distinct receptors: V1a and V1b receptors, which induce phospholipase-Cbeta (PLCbeta) activation and Ca2+ mobilization; and V2 receptors, which are coupled to adenylyl cyclase. V1a and V1b receptors are also present in neurons. In particular, hypoglossal (XII) and facial (VII) motoneurons are excited following vasopressin-V1a receptor binding. The aim of the present study was double: (i) to determine whether V1b receptors contribute to the excitatory effect of vasopressin in XII and VII motoneurons; and (ii) to establish whether the action of vasopressin on motoneurons is mediated by Ca2+ signalling. Patch-clamp recordings were performed in brainstem slices of young rats. Vasopressin depolarized the membrane or generated an inward current. By contrast, [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP), a V1b agonist, had no effect. The action of vasopressin was suppressed by Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2, a V1a antagonist, but not by SSR149415, a V1b antagonist. Thus, the vasopressin-induced excitation of brainstem motoneurons was exclusively mediated by V1a receptors. Light microscopic autoradiography failed to detect V1b binding sites in the facial nucleus. In motoneurons loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, the effect of vasopressin was suppressed, indicating that neuronal V1a receptors are G-protein-coupled. Intracellular Ca2+ chelation suppressed a Ca2+-activated potassium current, but did not affect the vasopressin-evoked current. H7 and GF109203, inhibitors of protein kinase C, were without effect on the vasopressin-induced excitation. U73122 and D609, PLCbeta inhibitors, were also without effect. Thus, excitation of brainstem motoneurons by V1a receptor activation is probably mediated by a second messenger distinct from that associated with peripheral V1a receptors.

Radioprotection of the mice parotid gland by isoproterenol: study on morphometry of secretory granules and on autoradiography

The aim of the present study was to investigate the effect of radiosensitivity on acinar cells in the parotid gland when the secretory granules were released. The parotid glands of mice were exposed to 10 Gy of X-radiation when the acinar cells were degranulated with isoproterenol (IPR). Three days later, morphological images and number and area of secretory granules within the acinar cells in the parotid glands were obtained and light microscope autoradiography (LMARG) was performed using 3H-leucine. The light microscopy images showed a disorderly arrangement and pycnosis of acinar cells and cellular atrophy in irradiated groups. The changes were milder in IPR-administered groups than in non-IPR-administered groups. The number of secretory granules in irradiated groups, which included both IPR-administered and non-IPR-administered sets, was significantly less than that in nonirradiated groups. The number of silver grains within acinar cells obtained by LMARG in the non-IPR-administered set of irradiated groups was significantly lower than that in the nonirradiated group or the IPR-administered set after 30 min of radioisotope administration, and it was significantly higher than that of the nonirradiated group after 240 min. When the secretory granules of acinar cells in mouse parotid gland were degranulated by isoproterenol, alleviation of the effects of radiation exposure on morphological change as well as the ingestion and egestion of secretory substances were indicated.

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: A light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1–M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1 > M2 > M4 > M3 > M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3–M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.

The existence of a linear system consisting of sympathetic endings in rat skin

In a previous pilot study we suggested the novel notion that the catecholaminergic sympathetic nerve endings are non-homogeneously distributed in the rat skin. In the present study we have utilized several independent methods to determine the in vivo distribution of catecholamine-containing fibers in rat skin. Using whole body macro-autoradiography with an iodine125-labeled tyrosine, we localized the distribution of iodine-125-catecholamine in rat skin. The images on the film showed various pairs of symmetrical linear arrays running from the head through the back and to the hind limbs of the animal that we arbitrarily termed sympathetic substance lines (SSLs). The distribution of catecholamine in rat skin was also visualized by light microscopy autoradiography with tritiated tyrosine. The majority of silver grains in the sections were located among hair follicles along a band or zone. Furthermore, a modified sucrose-phosphate-glyoxilic acid (SPG) method was adapted to observe sympathetic fibers in the skin sections. Dense clusters of fluorescent nerve fibers in correspondence of arrector pili muscles (AP muscles) were located along lengthwise lines of the body, in a pattern coinciding with the linear arrays identified by macro-autoradiography. We concluded that concentrated clusters of noradrenergic nerve fibers innervate AP muscles and form a longitudinal linear system in the whole skin. These results are discussed in terms of physiological functions associated with hair follicles, sensory signal pathways and Meridians in Chinese traditional medicine.