A simple standard technique for labyrinthectomy in the rat: A methodical communication with a detailed description of the surgical process.

Aims Labyrinthectomized rats are suitable models to test consequences of vestibular lesion and are widely used to study neural plasticity. We describe a combined microsurgical-chemical technique that can be routinely performed with minimum damage. Methods Caudal leaflet of the parotis is elevated. The tendinous fascia covering the bulla is opened frontally from the sternomastoid muscle's tendon while sparing facial nerve branches. A 4 mm diameter hole is drilled into the bulla's hind lower lateral wall to open the common (in rodents) mastoid-tympanic cavity. The cochlear crista (promontory) at the lower posterior part of its medial wall is identified as a bony prominence. A 1 mm diameter hole is drilled into its lower part. The perilymphatic/endolymphatic fluids with tissue debris of the Corti organ are suctioned. Ethanol is injected into the hole. Finally, 10 µL of sodium arsenite solution (50 µM/mL) is pumped into the labyrinth and left in place for 15 min. Simple closure in two layers (fascia and skin) is sufficient. Results and conclusion All rats had neurological symptoms specific for labyrinthectomy (muscle tone, body position, rotatory movements, nystagmus, central deafness). Otherwise, their behavior was unaffected, drinking and eating normally. After a few days, they learned to balance relying on visual and somatic stimuli (neuroplasticity).

Regional differences in statistical geometry of endothelial dense granules in human extremity veins.

OBJECTIVES: Leg and arm human veins are exposed to different gravitational stresses. We investigated if there is difference in the amount and geometry of secretory vesicles in their endothelium. METHODS: Superficial small vein segments were removed during vascular operations for electromicroscopic analysis. Vesicular area/total endothelial cross-sectional area was determined by computer-based morphometry. Long and short axes of granule cross sections were measured by image analyzing software. RESULTS: Vesicular density in all samples was 2.26 ± 0.34%. There was no significant difference between the vesicular densities of upper extremity and leg. The shape of the vesicles was more frequently elongated in leg than in arm sections (p < 0.01). CONCLUSIONS: The density of the vesicles does not depend on vascular region or orthostatic load. Ellipticity of these granules is significantly different in areas exposed to different gravitational stresses. This might contribute to the differences of thrombotic and hemodynamic properties of leg and upper body veins.

Vestibular control of intermediate- and long-term cardiovascular responses to experimental orthostasis.

Sustained orthostasis elicits the elevation of arterial blood pressure (BP) via sympathetic activation in conscious Wistar rats for at least 2 hours. We tested the hypothesis whether vestibular apparatus plays a role in BP and heart rate (HR) control in response to prolonged gravitational stress. BP and HR responses to 45 degrees head-up for either 2 or 24 hours were monitored by telemetry. Vestibular lesions (VL) were performed by a modified microsurgical-chemical technique. Horizontal BP and HR were not influenced by VL preceding 2-hour tilt. VL abolished the sustained 2-hour BP response to head-up tilt (8.3+/-0.9 mm Hg relative to horizontal values) while suppressed HR transiently only. VL eliminated diurnal BP fluctuations and decreased HR in horizontal position for 24 hours. Head-up tilt for 24 hours increased BP and HR progressively in intact animals, raising their daily average value by 5.6+/-0.7 mm Hg and 22.2+/-6 BPM, respectively. VL resulted in an initial BP rise followed by progressive BP reduction in response to long-term head-up tilt (4+/-2.2 mm Hg) without eliminating the tachycardia (34.4+/-5.4 BPM). Thus, blockade of labyrinthine inputs attenuates the BP responses elicited by both intermediate and long-term gravitational stress of orthostatic type. However, other sensory inputs derived from non-vestibular cues (e.g. proprioceptive, visual, visceral, cutaneous etc.) seem to be effective enough to maintain BP normal.

Does long-term experimental antiorthostasis lead to cardiovascular deconditioning in the rat?

Microgravity or simulated microgravity induces acute and chronic cardiovascular responses, whose mechanism is pivotal for understanding of physiological adaptation and pathophysiological consequences. We investigated hemodynamic responses of conscious Wistar rats to 45? head-down tilt (HDT) for 7 days. Arterial blood pressure (BP) was recorded by telemetry. Heart rate (HR), spectral properties and the spontaneous baroreflex sensitivity (sBRS) were calculated. Head-up tilt (HUT) was applied for 2 h before and after HDT to assess the degree of any possible cardiovascular deconditioning. Horizontal control BP and HR were 112.5+/-2.8 mmHg and 344.7+/-10 bpm, respectively. HDT elicited an elevation in BP and HR by 8.3 % and 8.8 %, respectively, in less than 1 h. These elevations in BP and HR were maintained for 2 and 3 days, respectively, and then normalized. Heart rate variability was unchanged, while sBRS was permanently reduced from the beginning of HDT (1.01+/-0.08 vs. 0.74+/-0.05 ms/mmHg). HUT tests before and after HDT resulted in BP elevations (6.9 vs. 11.6 %) and sBRS reduction (0.44 vs. 0.37 ms/mmHg), respectively. The pressor response during the post-HDT HUT test was accompanied by tachycardia (13.7 %). In conclusion, chronic HDT does not lead to symptoms of cardiovascular deconditioning. However the depressed sBRS and tachycardic response seen during the post-HDT HUT test may indicate disturbances in cardiovascular control.

Increased diameter and enhanced myogenic response of saphenous vein induced by two-week experimental orthostasis are reversible.

Previously, increased diameter and enhanced myogenic tone were seen after 2-week 45o head-up (HUT2) in the rat. We studied the reversibility and the effect of extended tilt on this phenomenon using two experimental groups: HUT2 plus 2-week horizontal (HUT2HOR2), and 4-week tilting (HUT4). 4-weeks in normal cages (NC4) served as control. Diameter of saphenous vein (SV) in 2-20 mm Hg pressure range, wall and media thickness, endothelial and smooth muscle cell densities, and cell proliferation were measured. The diameter of SV from HUT4 was significantly larger compared with HUT2HOR2 or NC4 within the whole pressure range both in Krebs-Ringer (870.4+/-21.3 vs. 778.2+/-24.9 and 771.6+/-28.1 microm at 10 mm Hg, respectively) and in Ca(2+)-free solution. Myogenic and norepinephrine-induced vascular tone, wall and media thickness did not differ among the three groups. Endothelial cell density decreased in HUT4 (10.7+/-1.2) vs. HUT2HOR2 (15.1+/-1.0) and NC4 (15.3+/-0.6), while that of smooth muscle was unchanged. No cell proliferation marker was seen. In conclusion, both increased diameter and enhanced myogenic tone of SV seen in HUT2 proved to be reversible. HUT4 resulted in increased SV diameter, similarly to HUT2, however, vascular tone was not amplified. This suggests that a prolonged orthostatic load may readjust the function of smooth muscle.

Does chronic experimental head-down tilt alter intramural innervation density of limb blood vessels?

Earlier, substantial increases in the intramural sympathetic innervation density of rat hind-limb blood vessels were found after 2 weeks of experimental orthostasis with tubular 45 degrees head-up tilt cages. In the present study, we presumed that chronic head-down tilting induces opposite changes in the innervation density. Tilted rats were kept 45 degrees head-down in long tubular cages for either 2 or 4 weeks (HDT2, HDT4), and the control animals were maintained in horizontal tilt cages for the same period (HOR2, HOR4). Segments of the saphenous and brachial veins and arteries were used for quantitative electron microscopic examinations. Intramural innervation density was defined by nerve terminal density (NTD) and synaptic microvesicle count (SVC) within the vascular adventitia. Neither HDT2 nor HDT4 resulted in a decrease of NTD or SVC of the saphenous and brachial veins or arteries; instead, a tendency to increase was observed in some cases. Thus in contrast to the large increases we found earlier in hind-limb vascular innervation density after 2 weeks of head-up tilting, head-down tilting of the same duration-or even twice as long-did not decrease the adventitial innervation density in our model. We assume that the quasi-free locomotor exercise the tilted animals in the long tubular cages were allowed may counteract a possible suppressive effect of chronic head-down tilt on hind-limb vascular innervation density.

Remodeling of the rat saphenous vein network in response to long-term gravitational load.

Our main objective was to test whether chronic orthostatic body position induces network changes in the saphenous vein superficial tributary system of the rat. Fourteen male Sprague-Dawley rats were kept in tilted tube cages (45-degree head-up position) for two weeks to induce chronic gravitational load to their leg veins. Ten animals housed in normal cages and four animals kept in horizontally positioned tube cages served as controls. The whole superficial network of the left saphenous vein was microprepared surgically under anesthesia, superfused with saline and observed under a videomicroscope, while normal flow and pressure were maintained in the lumen. Branching angles, lengths of venous segments and their diameters were measured offline from digitized images using special image-analyzing software. Several branching angles at the popliteal confluence were significantly reduced by 12.5-15.8 %. The in vivo diameter of the main branch (936+/-34 vs. 805+/-44 microm) and of one of the popliteal tributaries (776+/-38 vs. 635+/-36 microm) increased (p<0.05), comparing vessels from tilted animals with those from normal controls. Maintaining the animals in horizontal tube cages did not induce the above alterations. The increased diameters and reduced branching angles of the saphenous vein network observed are adaptive responses of the venous network to a long-term gravitational load.

Influence of long-term head-down body position on innervation density in extremity blood vessels.

The aim of the present study was to quantitate and compare the density of nerve terminals (NTD), as well as of their synaptic vesicle population (SyVD) in saphenous and brachial vein and artery, obtained from rats maintained in the horizontal or head-down tilted (HDT) position for two weeks. The same technique was applied as that for the head-up tilt study.

Axial stretching of extremity artery induces reversible hyperpolarization of smooth muscle cell membrane in vivo.

Circumferential stretch due to increases in pressure induces vascular smooth muscle cell depolarization and contraction known as the myogenic response. The aim of this study was to determine the in vivo effects of axial-longitudinal stretch of the rat saphenous artery (SA) on smooth muscle membrane potential (Em) and on external diameter. Consecutive elongations of the SA were carried out from resting length (L0) in 10% increments up to 140% L0 while changes in membrane potential and diameter were determined in intact and de-endothelized vessels. Axial stretching resulted in a small initial depolarization at 120% of L0 followed by a progressive 20 to 33% hyperpolarizaion of vascular smooth muscle between 130% and 140% of L0. At 140%, an average maximal 10.6 mV reversible hyperpolarization was measured compared to -41.2 +/- 0.49 mV Em at 100% L0. De-endothelialization completely eliminated the hyperpolarization to axial stretching and augmented the reduction of diameter beyond 120% L0. These results indicate that arteries have a mechanism to protect them from vasospasm that could otherwise occur with movements of the extremities.

Validity of viscoelastic models of blood vessel wall.

Quantitative description of biomechanical behaviour of the blood vessel wall is needed for understanding normal and pathologic functions of the vascular system. In this study we investigated the stress relaxation of blood vessels. Strips were cut from the vessel wall and they were stretched in a stepwise manner until they tore. The mechanical stress (approximately 10(5)-10(6) Pa) induced in the strips was determined. This showed an initial steep rise and then a slow, near exponential decay (stress relaxation) after each step of stretch. For the mathematical description we used two technical models (the Maxwell and the Kelvin model--they are built of two elastic elements and of one viscous element), as well as a one-dimensional continuum mechanical model. The moduli of the models were identified with a curve fitting method. Our aim was to compare these models in order to elucidate, which one describes the rheological behaviour of the blood vessel more correctly. The results of our analysis show that the corresponding moduli diverge from each other in the Maxwell and Kelvin models: that is, they depend on the mode of the assumed connection between the mechanical units. In contrast, moduli of the continuous model are unambiguous. We conclude that the continuous model properly describes the stress relaxation of the vascular wall. Its advantage over the technical models is that moduli describing the viscoelastic behaviour of the tissue do not depend on the not wholly well-known structure of the wall components.