Robert M. Klein

Autoradiographic assessment of 3H-proline uptake by osteoblasts following guanethidine-induced sympathectomy in the rat

SummarySympathectomy was carried out in rats by injections of guanethidine-sulfate from birth to 14 days of age. At 45 days of age, the activity of osteoblastic cells was monitored by 3H-proline autoradiography. Effectiveness of sympathectomy was verified by light-microscopic examination of superior cervical and celiac ganglia. Grain counts over periosteal osteoblasts of the femoral diaphysis and osteoblasts mesial to the first molar in the mandible demonstrated a significantly reduced uptake of 3H-proline in the sympathectomized rats. The data provide direct evidence of sympathetic influence on osteoblastic activity and suggest that sympathectomy may result in the loss of a trophic influence which is important in the regulation of osteogenesis.

Pattern of crypt cell proliferation in the pre- and post-closure ileum of the neonatal rat: effects of sympathectomy.

SummaryThe present experiments were designed to investigate ileal crypt cell division before and after neonatal closure to macromolecular absorption by measurements of mitotic index, labelling index, colchicine-induced estimation of mitotic rate (mitoses/cell/hour) and crypt depth and villus height on postnatal days 15–23. In addition, the effects of the sympathetic nervous system on crypt cell proliferation were analyzed by guanethidine-induced sympathectomy. Guanethidine treatment resulted in at least a 70 % reduction in superior cervical and coeliac perikarya at 15 days after birth. All cell proliferative indices demonstrated a rather constant rate of cell division in the ileal epithelium between 15 and 17 days after birth with a sudden burst of mitotic activity on day 18. The mitotic rate of control rats increased from day 18 to 23 with 3-week-old rat demonstrating a faster rate of ileal crypt cell proliferation than adult rats. The ileal crypt depth more than doubled between 15 and 23 days while the height of the villus column increased slowly but steadily (36 %) during the period of this study. Cell division is inhibited by guanethidine-induced sympathectomy although there is still an acceleration of mitosis in the post-closure period. The relationship of sympathectomy and ileal crypt cell proliferation is discussed and compared to hormonal effects on closure and related developmental events.

Autoradiographic analysis of cell proliferation and protein synthesis in the pulmonary trunk of rats during the early development of hypoxia-induced pulmonary hypertension

The results of this study indicate that both cell proliferation and increased synthesis of extracellular matrix protein contribute to hypertrophy of the rat pulmonary trunk during the early development of hypoxia-induced pulmonary hypertension. As determined by autoradiography after 3H-thymidine injection, pulmonary hypertension results in increased labelling in all cell compartments of the pulmonary trunk wall, the most dramatic response occurring in the adventitia following 3 days hypoxic exposure. Autoradiography also demonstrated differences in the degree of incorporation of 3H-proline into extracellular protein between hypoxic (3 and 21 days) and control rats. The major focus of 3H-proline incorporation shifted from the adventitia at 3 days to the tunica media at 21 days, although incorporation was significantly higher at 3 compared to 21 days in all wall compartments. The patterns of hyperplasia and matrix protein synthesis in the extrapulmonary arteries of the rat, as reported here, are distinctly different from those seen in many large elastic arteries during development of systemic hypertension. For example, the hyperplastic response of arterial vessels follows a similar temporal sequence in pulmonary and systemic hypertension. However, the adventitia is the region of the pulmonary trunk with highest cell proliferation in pulmonary hypertension while the media is most affected by systemic hypertension. The relevance of the changing patterns of cell proliferation and protein synthesis in the wall of the pulmonary trunk of chronically hypoxic rats to the structural and biochemical properties of this vessel during the early development of pulmonary hypertension is discussed.

TRITIATED THYMIDINE AUTORADIOGRAPHIC STUDY OF THE EFFECTS OF INFERIOR ALVEOLAR NERVE RESECTION ON THE PROLIFERATIVE COMPARTMENTS OF THE MOUSE INCISOR FORMATIVE TISSUES

Summary-At 15, 30 and 60 days after denervation, 10 mice were injected (5 controls, 5 denervated) at each time period with 0.5 @i/gm of C3H]-thymidine. Denervated teeth, after 15 and 30 days, were thinner, shorter and narrower with a chalky-white appearance; by 60 days, the teeth returned to their normal predenervated appearance. The responses of the proliferative compartments of the mouse incisor varied with time after denervation, and depended on whether comparison was made to sham controls or the control side of a denervated mouse. The pre-odontoblasts were most stable in terms of C3H]-thymidine labelling index with a significant decrease only at 60 days after surgery. This decrease may be due to an alteration of the inductive influence of the inner enamel epithelium and the nerve resection. The effects of nerve. resection on the inner enamel epithelium were a reduction in the labelling index on both control and clenervated sides in comparisons to sham controls and a decrease in labelling index of the denervated side when compared to contralateral control. Significant differences between the control and denervated sides of experimental mice were evident 15 days after denervation in the outer enamel epithelium, inner enamel epithelium, and pulp fibroblasts. Indirect effects (i.e. chipping of t’he teeth) may mask neurotrophic influences. There also appears to be a homeostatic or compensatory mechanism for attenuation of the effects of denervation in continuously erupting teeth. The effects at the early time periods emphasize the need for a study of neurotrophic effec1.s on the cellular compartments of the tooth in the period immediately following denervation.

EFFECTS OF GUANETHIDINE-INDUCED SYMPATHECTOMY ON CELL PROLIFERATION IN THE PROGENITIVE COMPARTMENTS OF THE NEONATAL MOUSE INCISOR

Summary-Treatment of mice with guanethidine sulphate every 48 h from birth until 14 days produced a 64.2 and 68.0 per cent reduction in perikarya of the superior cervical and coeliac ganglia, respectively, at 15 days after birth and 78.9 and 81.3 per cent at 30 days. The growth rate of mice between 21 and 30 days after birth was significantly reduced. At 15 and 30 days after birth, C3H]-thymidine was injected into control and sympathectomized mice, and labelling indices for the inner enamel epithelium, outer enamel epithelium, stratum intermedium, stellate reticulum, odontoblasts, and pulp and periodontal ligament fibroblasts determined from autoradiographs. A statistically significant decline in labelhng index was found in the inner enamel epithelium, stellate reticulum, and odontoblast cell populations at 30 days as compared to 15 days in controls. Sympathectomized mice demonstrated a significant decline in labelling index in the inner enamel epithelium, stratum intermedium, odontoblasts, and pulp fibroblasts at 30 days as compared to 15 days. The sympathetic nervous system, therefore, has only a minimal role in regulation in incisor eruption and cellular proliferation. The effect of sympathectomy on the stratum intermedium and stellate reticulum may be due to increased tissue pressure resulting from loss of vasoconstrictive sympathetic control.

β-adrenergic drug induced hyperplasia in the immature rat parotid and submandibular glands

In this study the proliferative response of rat parotid (PRG) and submandibular (SMG) gland acinar cells to beta-adrenergic stimulation with Isoproterenol (a non-selective beta-agonist) or terbutaline (a relatively selective beta 2-agonist) was determined during the 4th-5th postnatal weeks in intact and guanethidine-sympathectomized (Sx) rats. Rats were divided into 2 age groups (21 and 28 days) and 6 experimental groups (control-intact (C), guanethidine-treated (Sx), intact + TER (C + TER), intact + Isoproterenol (C + IPR), guanethidine-treated + TER (Sx + TER), and guanethidine-treated + isoproterenol (Sx + IPR]. Intact and Sx rats were treated with either IPR or TER for 3 days (days 21, 22, and 23 or days 28, 29 and 30). On day 24 or 31 all rats were injected with [3H]-thymidine and killed 1 h later. PRG of Sx and C + TER rats showed no significant differences from controls in wet weight while SMG from Sx rats demonstrated a reduced wet weight compared to C rats. [3H]Thymidine labelling index and mitotic index were not significantly different in Sx and C + TER rats in either PRG or SMG. C + IPR rats showed increased wet weight, labelling and mitotic index compared to controls (P less than 0.01). Sx + IPR increased values over controls (P less than 0.01) and above C + IPR values (P less than 0.01). Sx + TER values were significantly higher than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Synthesis in the Rat Pulmonary Trunk during the Early Development of Hypoxia-induced Pulmonary Hypertension

The purpose of this study was to determine the temporal alterations in protein synthesis and accumulation in the rat pulmonary trunk during the early development of hypoxia-induced pulmonary hypertension and to correlate these results with the pattern of development of polycythemia, right ventricular hypertrophy (RVH) and increased right ventricular pressure (RVP). In vitro synthesis of collagen and noncollagen protein was determined in the pulmonary trunks (PT) of rats exposed to chronic hypobaric hypoxia (0.5 atm/380 Torr) for 3, 7, 10, 14 or 21 days and in respective control groups of pair-fed normoxic rats. In vitro collagen synthesis was increased 1,150% (p less than 0.01) in PT from rats exposed to hypoxia for 3 days compared to PT from normoxic rats. When duration of hypoxic exposure increased, the percentage increase in collagen synthesis in PT from hypoxic vs. normoxic rats declined but remained significantly elevated (340%, p less than 0.005) after 21 days. Comparison of in vitro synthesis of noncollagen protein demonstrated a similar pattern of alteration with increasing hypoxic exposure. Synthesis of noncollagen protein was increased 750% in PT from hypoxic rats compared to normoxic controls at 7 days (p less than 0.0005) and decreased in parallel with collagen synthesis until 21 days when the difference in mean noncollagen protein synthesis was no longer statistically significant. In contrast, the accumulation of protein in vitro during hypoxic exposure, as determined from measurement of absolute protein and hydroxyproline content, demonstrated a pattern of continued increase with hypoxic exposure. Absolute protein content (microgram protein/vessel) was increased 330% in PT from hypoxic rats after 3 days compared to controls (p less than 0.005). By 21 days, mean absolute protein content of hypoxic PT was increased 500% compared to controls (p less than 0.0005). Absolute hydroxyproline content became significantly elevated in PT from hypoxic rats compared to controls after 7 days (123%, p less than 0.005 and the difference increased to 135% at 21 days (p less than 0.025). From comparison of these results with the pattern of changes in established indicators of pulmonary hypertension, herein reproduced, it is deduced that increased protein synthesis is a very early response of the pulmonary trunk to the stimulus of increased arterial blood pressure. Protein synthesis then declines while accumulation increases in a manner which suggests that the newly synthesized protein alters the response of the vessel to the continuing stimulus. It is proposed that increased accumulation of protein, particularly collagen, decreases the distension of the vessel wall caused by increased transmural pressure.