Mesenteric lymph flow in adult and aged rats

Abstract

The objective of study was to evaluate the aging-associated changes, contractile characteristics of mesenteric lymphatic vessels (MLV), and lymph flow in vivo in male 9- and 24-mo-old Fischer-344 rats. Lymphatic diameter, contraction amplitude, contraction frequency, and fractional pump flow, lymph flow velocity, wall shear stress, and minute active wall shear stress load were determined in MLV in vivo before and after N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) application at 100 μM. The active pumping of the aged rat MLV in vivo was found to be severely depleted, predominantly through the aging-associated decrease in lymphatic contractile frequency. Such changes correlate with enlargement of aged MLV, which experienced much lower minute active shear stress load than adult vessels. At the same time, pumping in aged MLV in vivo may be rapidly increased back to levels of adult vessels predominantly through the increase in contraction frequency induced by nitric oxide (NO) elimination. Findings support the idea that in aged tissues surrounding the aged MLV, the additional source of some yet unlinked lymphatic contraction-stimulatory metabolites is counterbalanced or blocked by NO release. The comparative analysis of the control data obtained from experiments with both adult and aged MLV in vivo and from isolated vessel-based studies clearly demonstrated that ex vivo isolated lymphatic vessels exhibit identical contractile characteristics to lymphatic vessels in vivo.

Fig. 1.

A: schematic of the imaging setup showing the animal board with an observation chamber under the objective lens. Data from the high-speed camera were downloaded to the processing computer at the end of each acquisition cycle. B: schematic of the principles of the data processing techniques used. Left: cell tracking principle used to measure white blood cell velocity. The Δ corresponds to the displacement of the cells between 2 frames and Δt is the time period separating the 2 frames. Right: diameter tracking technique where two windows with selected sites of vessel walls were tracked over time and the distance (d) between the windows (W₁ and W₂) at any acquired time point (t) defines the diameter D. Details of the approach are provided in the methods.

Fig. 2.

Representative diameter tracings of mesenteric lymphatic vessels (MLV) in vivo obtained in 9-mo rat overlapped with corresponding lymph flow velocity data points (A and B), or with wall shear stress data points (C and D) recorded under control conditions (A and C), and after nonspecific nitric oxide synthase (NOS) blockade induced by 100 μM of N^ω-nitro-l-arginine methyl ester (l-NAME; B and D).

Fig. 3.

Representative diameter tracings of MLV in vivo obtained in 24-mo rat overlapped with corresponding lymph flow velocity data points (A and B), or with wall shear stress data points (C and D) recorded under control conditions (A and C), and after nonspecific NOS blockade induced by 100 μM of l-NAME (B and D).

Fig. 4.

Aging-associated alterations in the parameters of the contractile activity of the 9- and 24-mo rat mesenteric vessels in vivo under control conditions and after nonspecific NOS blockade induced by 100 μM of l-NAME for 5 and 15 min. A–F: contractile activity parameters as noted on the vertical axis. Significant differences between active lymph pump parameters: ◊P ≤ 0.05, 9-mo control vs. 24-mo control and 9-mo l-NAME vs. 24-mo l-NAME; *P ≤ 0.05, 9-mo control vs. 9-mo l-NAME conditions and 24-mo control vs. 24-mo l-NAME conditions.

Fig. 5.

Aging-associated alterations in lymph flow velocity (A) and wall shear stress (B) in the 9- and 24-mo rat mesenteric vessels in vivo under control conditions and after nonspecific NOS blockade induced by 100 μM of l-NAME for 5 and 15 min. DV, diastolic lymph flow velocity; SV, systolic lymph flow velocity; D Sh S, diastolic wall shear stress; S Sh S, systolic wall shear stress; l-NAME 5 min and l-NAME 15 min, same parameters during the l-NAME (100 μM) administration within first 5 min and between 5 and 15 min, respectively. Significant differences between active lymph pump parameters: #P ≤ 0.05, 9-mo control vs. 24-mo control; *P ≤ 0.05, 9-mo control vs. 9-mo l-NAME conditions and 24-mo control vs. 24-mo l-NAME conditions.

Fig. 6.

Aging-associated alterations in the indexes of the active wall shear stress namely, active wall shear stress/dt (A) and active shear * frequency product (B) in the 9- and 24-mo rat mesenteric vessels in vivo under control conditions and after nonspecific NOS blockade induced by 100 μM of l-NAME for 5 and 15 min. Significant differences between active lymph pump parameters: ◊P ≤ 0.05, 9-mo control vs. 24-mo control; *P ≤ 0.05, 24-mo control vs. 24-mo l-NAME conditions.

Fig. 7.

Comparison of the parameters of the contractile activity of the adult 9- and 24-mo rat MLV in vivo vs. those parameters of the isolated rat MLV under control conditions and after nonspecific NOS blockade induced by 100 μM of l-NAME. A–C: contractile activity parameters as noted on the vertical axis. Control: parameters of contractility of MLV in vivo or in isolated MLV at intravascular pressures of 1 cm H₂O (Isolated P = 1) or at 3 cm H₂O (Isolated P = 3). Significant differences (P ≤ 0.05) between active lymph pump parameters: ◊P ≤ 0.05, 9-mo control vs. 24-mo control and 9-mo l-NAME vs. 24-mo l-NAME; *P ≤ 0.05, MLV in vivo vs. isolated MLV (for such comparisons the horizontal line adjacent to * demonstrates pairs of the contractile parameters where the significant differences were observed).