Dynamics of the functional activity of the lymph nodes under conditions of sympathectomy

By means of mechanotron 6MYIC and perfusion with physiological solution using the "running vesicle", dynamics of functional activity of the popliteal lymph nodes have been studied in 17 non-inbred dogs under conditions of unilateral sympathectomy for 1 week up to 5 months of the experiment. Dependence of the lymph node functional activity on the adrenomediator concentration level in their tissues has been determined by means of the microfluorometry method in the preparations treated after Falck-Hillarp method in E. M. Krokhina modification. Lymph nodes of intact dogs serve as a control. Unilateral sympathectomy of the lymph node is stated to produce an enhancement of the period for its filling and emptying with lymph, resulting from an increased level of the mediator in the fibrillar stroma at the expense of compensatory enhancement of functional activity of the nervous fibers of the contralateral origin. During the remote postoperative period the amplitude of the lymph node capsule fluctuations is stipulated by certain pathomorphological changes, produced by deficiency in concentration of catecholamines in the organ's parenchyma. As to the periodicity of the capsule fluctuations in the intact lymph nodes, it is limited with the cycle of 3-6 min.     

Catecholamine and serotonin content in the lymph nodes following deafferentation

Using Falck fluorescent technique in combination with microspectrofluorometry the measurement of catecholamine and 5-hydroxytriptamine level was performed in popliteal lymph nodes of normal dogs and 2 weeks after deafferentation. Maximal catecholamine and 5-hydroxytriptamine content was observed in adrenergic nerve fibers, elements of trabecular-elastic complex and monoaminocytes of medullary substance of intact animals. Biogenic amine level decreased deafferented limb lymph nodes. Monoamine imbalance according to divergent type was registered in a colateral limb, i. e. 5-hydroxytriptamine level was increased and catecholamine level was decreased.     

Degree of chromatin activity and the RNA level in lymphocytes of popliteal lymph nodes of dogs after administration of an antigen]

In the experiment performed on 127 dogs by means of cytospectrofluorometric analysis, using fluorochrome acridine orange in dynamics up to 1 year, changes in the level of chromatin activation and RNA content have been studied in lymphocytes of the germinative centers and the crown of lymphoid nodules, in the paracortical zone and medullary cords of the regional and contralateral popliteal lymph nodes, after subcutaneous injection of antigen (BCG vaccine, 0.2 mg/kg) into the lateral area of the foot of the left pelvic extremity. The immune response is accompanied with a periodical increase in the level of chromatin activation and RNA content in populations of lymphocytes in the regional and contralateral popliteal lymph nodes with maximum in 6 h, 3-7 days, 1-3 months after the antigen injection. The intensity of these processes has an unequal level in lymphoid cells of various structural components; it is higher in lymphocytes of the contralateral lymph node.     

Spread of herpes simplex virus in lymph nodes after experimental infection of mice

Herpes simplex virus was frequently isolated from ipsilateral popliteal lymph nodes after percutaneous inoculation of the dorsal face of the footpad, and from ipsi- and contralateral submandibular lymph nodes after percutaneous inoculation of the cheek or the orofacial area of mice. Virus was detected only on very rare occasion in nondraining lymph nodes (inguinal or axillary) or in contralateral popliteal lymph nodes, but was frequently isolated in contralateral lumbar lymph nodes after footpad inoculation. The presence of virus in lymph nodes paralleled or followed the invasion of ipsilateral sensory ganglia and was associated with dissemination of virus in contralateral sensory ganglia after unilateral inoculation. In older mice virus was detected only occasionally in lymph nodes and dissemination of virus in contralateral sensory ganglia was generally not observed. The results suggest that lymphatic spread may contribute to dissemination of virus in contralateral sensory ganglia after unilateral inoculation of mice.     

Adrenergic innervation of lymph nodes and the thoracic duct

By means of incubation of slices in 2% solution of glyoxylic acid distribution of adrenergic fibers in the rabbit lymph nodes and in the thoracic lymphatic duct has been studied. Adrenergic fibers get into parenchyma of the lymph nodes via two ways. The first--the perivascular, when the nervous fibers make a plexus and get into the node along the blood vessels, the second--diffuse nervous fibers get together with trabecules in between the lymphoid nodules. The distribution density of the adrenergic fibers is not the same in different groups of the lymph nodes. In the lumbar nodes it is the highest. In the lymph nodes of the cervical part the density of the sympathetic fibers is, as a rule, lower than in the lumbar, but higher than in the axillary nodes. The lowest density of th adrenergic fibers is in the mesenteric, superficial inguinal lymph nodes and in the lymph nodes, situating near the thoracic part of the aorta. In the lymphatic duct wall small amount of adrenergic fibers are revealed, they form a plexus, predominantly in the cranial part.     

An improved glyoxylic acid technique for the histochemical localization of catecholamines in brown adipose tissue

Summary A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

Dynamics of lymph node function in disordered innervation

A mechanotron 6MX1C was used to study lymphodynamic indices of the dog popliteal lymph nodes after cutting the ischiatic and femoral nerves in the left limb at 1 week, 3 and 5 months. The lymph nodes of the contralateral limb and the lymph nodes from intact dogs were used as controls. It has been demonstrated that denervation causes acceleration of the periods of the filling and emptying of the lymph nodes both in the denervated and contralateral limbs as compared with the initial period. The amplitude of fluctuations under maximal filling of the lymph nodes decreases significantly at the long-term postoperative periods. The lymphodynamic changes seem to be determined by pathomorphological alterations in lymph node tissues.     

Modulation of in situ murine neuroblastoma tumor growth by the adrenergic nervous system: differential response of clonal cell lines to chemical sympathectomy

The in situ growth characteristics of C-1300, N1E-115 and NS20Y murine neuroblastoma (MNB) tumor cell lines were compared in normal and sympathectomized A/J mice. Adrenergic nerve ablation was produced in neonatal mice by administration of 6-hydroxydopamine (6-OHDA) at a dose of 100 micrograms/gm body weight on post-natal days 4, 6, 8 and 10; controls received equivalent volumes of the vehicle solution (0.9% NaCl/0.1% Ascorbic Acid). All mice were inoculated subcutaneously with 10(6) viable MNB cells four to six weeks after treatment with 6-OHDA or vehicle. The growth rates of tumors produced by the adrenergic MNB cell lines, C-1300 and N1E-115, were significantly lower in sympathectomized mice when compared to control animals. In contrast, tumors induced by the cholinergic MNB cell line, NS20Y, grew at similar rates in both sympathectomized and control mice. All tumors obtained from control and sympathectomized mice regardless of whether they derived from cell lines characterized as cholinergic (NS20Y) or adrenergic (C-1300, N1E-115), contained both norepinephrine and dopamine. Depletion of adrenergic neurotransmitter in A/J mice was induced by administration of reserpine (5-10 micrograms/kg/day) beginning 30 days prior to implantation of the C-1300 MNB cell line and continuing until sacrifice of the animal. The effect of this treatment on organ and tumor catecholamine concentrations was confirmed by high-pressure liquid chromatography. Splenic catecholamine levels in reserpine-treated animals were reduced to 20% of controls as compared to 9% in the neonatally-sympathectomized group. However, there was no discernible effect on C-1300 MNB tumor growth in the reserpine-treated animals. C-1300 MNB tumor concentrations of nor-epinephrine and dopamine were significantly lower in the reserpine-treated animals than in controls. The suppression of tumor growth by adrenergic nerve ablation is selective for specific MNB tumor cell lines. An anatomically intact sympathetic nervous system appears to exert a greater influence than competency of adrenergic neuro-humoral transmission on MNB tumor growth. These data support the hypothesis that modulation of MNB tumor growth by the adrenergic nervous system is not mediated via catecholamines but may be modulated by endogenous growth factor(s).     

Morphofunctional restructuring of the nervous system of the lymphoid organs during electrostimulation of the hypothalamus

The intramural nervous apparatus of rabbit lymphoid organs was examined after 15 and 30 sessions of electric stimulation of the posterior hypothalamus. The function of nerve cells was evaluated by cytophotometry according to the content of catecholamines and acetylcholinesterase (ACE) activity. In the mesenteric lymph nodes, stimulation led to a decrease in the activity of both adrenergic and ACE-containing nerve cells. The spleen demonstrated a reduction in the catecholamine content in the perivascular nerve plexuses, with a simultaneous rise of the catecholamine content in the fibers located in the parenchyma, which evidences inhibition of the neurogenous mechanisms of vasomotor control and possible selective activation of the sympathetic mechanisms involved in the regulation of the immunogenesis. Thymic preparations impregnated with silver salts manifested high argyrophilia of the nerve cells, characteristic of the reactive phase of the destructive process. The problems of the hypothalamic effects on the blood supply and functions of the immune system organs are discussed.     

Ovarian sympathectomy in the guinea pig

Summary The role of ovarian adrenergic nerves in follicular growth was studied in prepubertal guinea pigs by determining the effect of sympathectomy on 1) follicle populations and 2) follicular development following exogenous gonadotropin administration. Selective unilateral ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian bursa on day 20 postpartum. The contralateral surgically closed ovarian bursa was injected with the vehicle used for 6-hydroxydopamine. On day 25, animals were injected with pregnant mare serum or saline followed by human chorionic gonadotropin or saline 48 h later. All animals were laparotomized on day 28 and blood from utero-ovarian veins was collected bilaterally for androstenedione determination. Ovaries were processed for morphometric analysis of follicles. The sympathectomized ovary in saline-injected animals had a significant decrease in preantral follicles (characterized by 2 layers of granulosa cells without antrum formation), an increase in 310–500 m diameter atretic follicles and an increase in follicles 700 um compared to the contralateral control ovary. There were no differences in androstenedione levels from the two sides, ovarian weights or the total number of follicles per ovary. Neither ovary had corpora lutea. The sympathectomized ovary in animals injected with gonadotropins was not different from the contralateral ovary in any of the parameters measured. Both control and sympathectomized ovaries had newly formed corpora lutea in response to the exogenous gonadotropins. These results suggest that ovarian adrenergic nerves normally participate in follicular development in the prepubertal guinea pig. However, exogenous gonadotropins may override neural influences on the prepubertal ovary.     

The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat

Summary The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme and immunohistochemical techniques at the light-microscopical level.The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes.Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla.The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found.Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.Abbreviations PLN popliteal lymph node - FDC follicular dendritic cell - IDC interdigitating cell - HEV high endothelial venule - TNP trinitrophenyl - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - GCPC germinal center precursor cell - sIg surface immunoglobulin - cIg cytoplasmic immunoglobulin     

Vascular bed of a lymph node and lymphodynamics after leg lengthening by the G. A. Ilizarov method

In the experiments performed on dogs, the right shin has been elongated, lymph movement has been studied in the extremity, as well as in the arteries of the popliteal and medial iliac lymph nodes. The lymph movement rate immediately after fracture decreases 6 times, and during the elongation process it gradually restores with normalization on the 14th day of distraction. The integrity of the arterial bed in the lymph node is not disturbed during the elongation process of the shin. The intraorganic bed of the regional lymph nodes reacts to the shin elongation by increasing diameter of the arteries and the number of the functioning vessels. The changes in the arterial bed demonstrate an acceleration of the blood stream in the lymph nodes under the elongation of the shin. After osteogenesis normalization in the lymph stream takes place.     

Ethanol-induced cardiac hypertrophy: effects of peripheral sympathectomy

The development of cardiac hypertrophy was examined in rats that had undergone sympathectomy with 6-hydroxydopamine. After 4 days, the rats were given severely intoxicating doses of ethanol or isocalorically paired quantities of maltose-dextrin by intubation at 8-h intervals up to 48 h. The ethanol and sugar intubations were applied in a nutritionally adequate, liquid diet mix. The extent of the peripheral sympathectomy was evident from the absence of detectable quantities of noradrenaline in hearts of animals injected with the neurotoxin and in the reduced levels of excreted noradrenaline. The adrenal medullary catecholamine contents of sympathectomized rats were unchanged in the absence of ethanol; there were reduced quantities of adrenaline in the medullae of rats given ethanol. The adrenal glands of rats given ethanol were larger than those from control animals. Urine samples from sympathectomized and control rats, given ethanol, displayed equivalent increases in excreted adrenaline and noradrenaline. Increases in relative cardiac weight were evident in hearts from sympathectomized animals after 4 days of sympathectomy, and this change reached significance in the hearts from 6-hydroxydopamine-treated rats after a further 2 days on the control diet. Hearts from animals exposed to ethanol showed a marked, rapid development of cardiomegaly; after 24 h there was an increased mass of some 17%, which was sustained over the remaining 24-h period. The proportion of cardiac protein did not differ in the large hearts from ethanol-treated animals and those from their controls, hence myocardial oedema could not account for the increase in weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of neonatal sympathectomy on the postnatal differentiation of the submandibular gland of the rat

Summary Right superior cervical sympathectomy was performed in one-day old rats. This operation had a small but definite effect on the postnatal development of the submandibular gland. The gland on the sympathectomized side weighed less and contained smaller amounts of DNA, RNA and protein than the contralateral intact gland. The postnatal development of acinar cells and granular convoluted ductal cells was retarded in the sympathectomized gland. The acinar cells which differentiated after the ganglionectomy were smaller than those in the contralateral intact gland and were filled with secretory granules but devoid of basal basophilia. The rate of cellular proliferation in the sympathectomized gland was, however, similar to that in the intact gland at various ages studied.Supported by Public Health Service Research Grant CA 17038 from the National Cancer Institute to Dr. T. Barka. The authors are indebted to Miss H. van der Noen and Mr. I. Borcsanyi for their assistance and to Dr. T. Barka for his suggestions regarding this paper     

Ovarian sympathectomy in the guinea pig

The influence of ovarian adrenergic nerves on follicular growth during the estrous cycle in the adult guinea pig was ascertained by comparing follicular development in control and chemically sympathectomized ovaries from the same animal. Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian membranous sac (bursa) on day 2 of the cycle (day 1 = day of estrus). The contralateral surgically closed ovarian bursa was injected with solvent used for 6-hydroxydopamine. Animals were laparotomized on days 5, 10 and 14 of the cycle. Blood from the utero-ovarian vein was collected bilaterally for measurement of progesterone and androstenedione. The ovaries were processed for histologic examination, and the number of follicles in each ovary was analyzed morphometrically. Sympathectomy on day 2 caused a decrease in healthy preovulatory follicles (greater than 700 micron diameter) on day 10 of the cycle. There were no differences in ovarian weights or the total number of follicles per ovary at this time. On days 5 and 14 of the cycle, there were no differences in ovarian weights, total number of follicles per ovary or follicles in any size classification. Sympathectomy did not alter progesterone levels in the utero- ovarian vein as compared to contralateral control levels. From control ovaries, there was a significant increase in progesterone in the blood of the utero-ovarian vein on day 10 but venous levels of progesterone from sympathectomized ovaries were not significantly different at any day of the cycle. In the venous effluent from sympathectomized ovaries, androstenedione was elevated at day 5 compared to days 10 and 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age changes in rat vasomotor reflexes and sympathetic neuron ultrastructure following chemical sympathectomy]

Prolonged administration of guanethidine (20 mg/kg) to newborn rats caused a marked reduction in the number of cells in stellate ganglia. The administration of guanethidine for 14 days decreased the amount of cells to 30% of the normal (partial sympathectomy), and for 28 days--to 0.5% (complete sympathectomy). At the age of two months the blood pressure pressor reflexes to asphyxia and femoral nerve stimulation were absent in both groups of the sympathectomized animals. These responses, however, were restored in the partially sympathectomized animals at the age of four months. No restoration took place in the completely sympathectomized animals. The electron microscopic studies of neurons in the partially sympathectomized animals showed the presence of a great number of neurofibrils. According to literature data this fact was typical of cells in which an active growth of axon fibers took place.     

THE INFLUENCE OF ADRENORECEPTOR ACTIVITY ON CRYPT CELL PROLIFERATION IN THE RAT JEJUNUM

The influence of adrenergic stimulation, adrenergic blockade and sympathectomy on crypt cell cycle time and mitotic time in rat jejunum was studied. Alpha adrenergic stimulation by noradrenaline was found to shorten both cell cycle and mitotic times, whereas beta adrenergic stimulation by adrenaline or isoprenaline was found to prolong both cell cycle and mitotic times. Conversely, alpha adrenergic blockade by phentolamine prolonged cell cycle time and beta adrenergic blockade by propranolol or practolol shortened cell cycle time but not mitotic time. Both chemical and surgical sympathectomy inhibited crypt cell proliferation. Chemically sympathectomized animals manifested supersensitivity to exogenous noradrenaline.     

Cryptococcal lymphadenitis in a dog.

We describe a case of canine crytococcosis, the clinical symptoms were: feverish syndrome, vomiting and diarrhoeas and bilateral lymphadenitis in superficial lymph nodes. Microbiology and histopathology study of popliteal lymph node biopsy demonstrated the presence of round yeasts of some 3 microm of diameter, which we identified as Cryptococcus neoformans. Thirty months after suspending the medication the dog returned to the surgery; the dog was very thin and it had nervous symptoms; the owners decided it upon euthanasia. After carrying out the necropsy we take samples for their microbiology and histopathology study, both techniques detect to C. neoformans in marrow, CNS tissue and lymph nodes.     

Lymphocyte migration during the development of regional lymph node anergy in experimental tumor growth

The development of lymph node anergy in Wistar rats to growing Walker carcinoma 256 was studied in vitro using the 51Cr-release cytotoxicity assay. Cell-mediated cytotoxicity to the tumor peaked in draining lymph nodes 11 days after tumor transplantation. By 14 days, the regional lymph node had become anergic to the tumor at a time when cell-mediated cytotoxicity was still increasing in the more distal contralateral lymph node. Lymphocyte migration into resting, cytotoxic, and anergic lymph nodes was analyzed to determine if altered cell migration into the regional lymph node was associated with the development of anergy. Lymphocyte migration was found to be enhanced in both cytotoxic and anergic regional lymph nodes of tumor-bearing animals. It is concluded that lymph node anergy in this experimental tumor system is not related to changes in lymphocyte migration patterns; rather, it is the result of alterations in the microenvironment of the lymph node which prevents the expression of cytotoxic effector cells.