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Blood Flow and Interstitial Fluid Pressure in the Rat Submandibular Gland during Changes in PerfusionDepartment of Physiology, Jonas Lies Vei 91, University of Bergen, N-5009 Bergen, Norway; Correspondence: * corresponding author, ellen.berggreen{at}fys.uib.no
The submandibular gland is a cell-rich encapsulated organ with high transport of fluid through the interstitial space during salivation. We hypothesized that the gland is a low-compliant tissue, i.e., that a modest increase in fluid volume will produce a rise in interstitial fluid pressure (IFP) counteracting fluid filtration into the interstitium. To test this hypothesis, we measured IFP with micropipettes and glandular blood flow (GBF) with a laser-Doppler flowmeter during changes in perfusion. Clamping of the carotid artery or the jugular vein, or electrical stimulation of the sympathetic or parasympathetic nerve to the gland, induced changes in perfusion. Baseline IFP averaged 3.5 ± 0.5 mm Hg. Clamping of the artery reduced IFP and GBF (–56.5 ± 8.4% and –53.1 ± 6.4%, respectively), whereas clamping of the vein decreased GBF (–21.6 ± 14.3%) and increased IFP (141.2 ± 27.4%). Sympathetic nerve stimulation reduced both parameters (–86.9 ± 16.5% and –74.4 ± 7.0%, respectively). In contrast, stimulation of the parasympathetic nerve elicited an increase in GBF (133.2 ± 5.9%) and in IFP (173.3 ± 41.4%). Thus, changes in vascular volume led to concomitant changes in IFP consistent with low tissue compliance, a phenomenon of importance for fluid volume regulation.
Key Words: laser-Doppler • micropuncture technique • nerve stimulation
During parasympathetic stimulation, salivary glands can increase their salivation from near zero to 700 µL/min/g and blood flow from 0.6 mL/min/g to 13 mL/min/g (Mann et al., 1979). The high flow of saliva and blood can be sustained and requires a high transport rate of fluid from the capillaries through the interstitial space, before secretion via the glandular epithelium. The interstitial space, in addition to being a transport medium, also acts as a fluid reservoir, and the interstitial fluid volume is regulated through changes in hydrostatic pressure and colloid osmotic pressure in the actual tissue. The forces balancing the volume flux across the capillary follow the Starling principle: Jv = CFC [(Pc-IFP) –  (COPp – COPi)] - L, where Jv = net flow across capillary, CFC = capillary filtration coefficient, Pc = capillary hydrostatic pressure, IFP = interstitial fluid pressure, = osmotic reflection coefficient to plasma proteins, COPp and COPi = colloid osmotic pressure in plasma and interstitium, respectively, and L = lymph flow. Most studies on the regulation of fluid flux in salivary glands have concentrated on transport across the glandular and vascular endothelium. Since secretion of saliva involves the transport of fluid across three main barriers (the vascular endothelium, the interstitial space, and the glandular epithelium), the role of the interstitial tissue should not be overlooked. In salivary glands, blood flow as well as salivary flow is under autonomic nervous control, and both parasympathetic and sympathetic nerves are involved. In view of the dramatic changes in fluid transport during nervous stimulation, it was considered important to measure blood flow and IFP simultaneously during induced changes in gland perfusion. We hypothesized that the encapsulated, cell-rich submandibular gland is a tissue with limited possibilities to expand where an increase in intravascular and/or interstitital fluid volume will give a concomitant rise in IFP, counteracting fluid filtration into the interstitium. If so, it is a tissue with low compliance, again of importance for gland fluid volume regulation. This study was therefore designed to test whether induced changes in gland perfusion caused immediate responses in IFP.
Female Wistar rats (range, 190–220 g, n = 15) were anesthetized with barbital sodium (50 mg/kg i.p.) and maintained with food and water ad libitum (2–3 mg/kg i.v.). The rats were studied while supine, and body temperature was kept at 37-38°C with a servocontrolled heating pad. A femoral vein was catheterized for supplemental anesthesia and a femoral artery for continuous systemic blood pressure (PA) recordings with a Gould pressure transducer and recorder. Dissection of the submandibular gland, the carotid artery, the jugular vein, the sympathetic trunk, or the lingual nerve (with parasympathetic fibers from chorda tympani) was performed, and the gland was placed in a cup for immobilization and covered with saline. To induce changes in glandular blood flow (GBF), we clamped the common carotid artery or the jugular vein for 1–2 min with a thin snare. After the artery or vein was clamped, the sympathetic trunk or the lingual nerve was stimulated electrically. In one rat, a submandibular gland was removed after the end of the experiment and was fixed in 4% paraformaldehyde with 0.2% picric acid in 0.1 M phosphate-buffered saline (pH 7.4). Frozen tissue was sectioned (40 µm) and stained with hematoxylin-eosin. The University of Bergen, under the supervision of the Norwegian Experimental Animal Board, approved the animal experiments.
Micropuncture Measurements of IFP
Blood Flow Recordings We used a Periflux Model 4001 Master laser Doppler flowmeter (Perimed KB, Tärfälla, Sweden,) equipped with a needleprobe PF 415:10 (fiber diameter 125 µm with 500-µm separation) to measure changes in GBF. The laser probe was positioned with a micromanipulator above the area for IFP recordings, and rotated to the position that gave the largest resting blood flow signal measured in arbitrary perfusion units (PU). By using a motility standard giving an output value of 50 PU, we carried out standard calibration of the instruments and fiber-optic probes according to the manufacturer’s specifications. Zero blood flow was determined as the value recorded with the probe positioned at the gland after heart arrest. The flowmeter set constant was 0.03 sec; upper and lower bandwidths were at 20 kHz and at 20 Hz, respectively.
Electrical Stimulation
Statistical Analyses
Baseline IFP in the submandibular glands averaged 3.53 ± 0.51 mm Hg and systemic blood pressure 95 ± 2.9 mm Hg (n = 15). After baseline registrations, GBF and IFP were recorded during and after clamping of the ipsilateral carotid artery. Clamping of the artery (n = 11) gave a concomitant drop in IFP as well as blood flow (-56.5 ± 8.4% and -53.1 ± 6.4% of baseline, respectively), whereas systemic blood pressure remained unchanged (WSRT) (Fig. 2  ). After the clamp was released, a brief hyperemia appeared concomitantly with a peak increase in IFP to 200-300% of baseline, followed by a gradual return to baseline of GBF and IFP within 2–3 min. When the ipsilateral vein was clamped (n = 6), a consistent fall from baseline blood flow (-34.3 ± 5.3%) was observed, followed by a prompt increase in IFP (141.2 ± 27.4%), without changes in systemic pressure (WSRT) (Fig. 2). The timing and effect on IFP (panel B) and GBF (panel C) are illustrated (Fig. 3).
As for the release of the arterial clamp, hyperemia followed venous clamp relief, and IFP and GBF were both elevated for 2–5 min. IFP remained elevated for a longer period than GBF, indicating that some fluid was filtered into the interstitial compartment during the clamping period, due to vascular distension and increased capillary pressure. Brief electrical stimulation caused immediate changes in blood flow and IFP. Sympathetic nerve stimulation (n = 5) induced a fall in both parameters (-63.72 ± 11.9% and -73.5 ± 18.5%, respectively) (Fig. 2  ). Parasympathetic stimulation (lingual nerve, n = 5) was followed by an increase in blood flow (135.3 ± 5.2%) and in IFP (173.3 ± 41.3%) (Figs. 2, 4) that returned to baseline immediately after stimulation ended.
A major finding in the present study was that the IFP is positive in the rat submandibular gland during control conditions (3.53 ± 0.51 mm Hg). Moreover, this pressure responds strongly to changes in vascular distension, suggesting that the gland behaves like an encapsulated organ with low interstitial and whole organ compliance. To our knowledge, these are the first recordings of IFP with the micropuncture technique, allowing us to measure rapid tissue pressure fluctuations in a salivary gland, and the present observations will have implications for further fluid balance studies in salivary glands.
In all experimental situations, changes in IFP immediately followed induced changes in blood flow. The alterations in IFP were all induced by blood volume changes in the gland, since they appeared before prospective interstitial fluid volume changes could have taken place, as exemplified by the response to venous stasis (Fig. 3 Interstitial hydrostatic pressure has previously been measured by the wick-in-needle technique in the rabbit submandibular gland, and was found to be about –1 mm Hg in resting conditions (Smaje, 1998). The difference might be due to the different experimental situation, species differences, and/or different techniques. The wick-in-needle technique is a more traumatic method compared with the micropuncture technique, because it requires implantation of a steel cannula into the tissue. During salivation induced by parasympathetic stimulation, IFP dropped to –2 mm Hg in the rabbit gland, indicating a net transport of fluid out of the interstitial tissue. The brief stimulation period with simultaneous IFP recording in our experimental set-up did not allow us to measure IFP during salivation. An advantage of the micropuncture technique is good time resolution, allowing us to observe an increase in IFP that appeared almost simultaneously with an increase in blood flow during, e.g., parasympathetic stimulation. Such rapid fluctuations cannot be unmasked with the wick-in-needle technique. Although the salivary gland was flushed repeatedly with 38°C saline, isolation of the gland might have caused a drop in temperature in the tissue. A temperature drop might have resulted in vasoconstriction and thereby caused a reduced fluid volume and IFP in the gland. We have measured IFP before and after gland isolation (unpublished data) and found no pressure difference in the two situations, and since we found no fall in blood flow or change in resting IFP in the time course of the experiments, this potential effect of isolation must be negligible.
Representative measurements of IFP with the micropuncture technique require extracellular location of the pipette tip. Since we cannot localize the tip precisely during measurements, we cannot rule out the possibility that the tip could be placed intracellularly in some recordings. Nevertheless, if some of our recordings should reflect intracellular pressure rather than IFP, the pressure will likely equal IFP, since hydrostatic pressure gradients are unlikely across the soft plasma membrane of cells (Macknight and Leaf, 1977). We also have to consider the possibility that some of our IFP measurements might reflect pressure in salivary ducts. Since the ducts in the area of IFP recordings have narrow lumens (Fig. 1 Taken together, our observations indicate that the submandibular gland has a relatively low compliance, a phenomenon of importance for fluid volume regulation. As a consequence, moderate changes in intravascular and/or interstitial fluid volume result in marked changes in interstitial fluid pressure, again counteracting fluid filtration into the interstitium.
We thank Aase R. Eriksen for technical assistance. This study was financed by a post-doctoral fellowship from the University of Bergen (to E. Berggreen) and by the Norwegian Research Council. Received for publication February 13, 2003. Revision received August 1, 2003. Accepted for publication August 27, 2003.
Journal of Dental Research, Vol. 82, No. 11,
899-902 (2003) This article has been cited by other articles:
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