Richard F. Potter

Capillary diameter and geometry in cardiac and skeletal muscle studied by means of corrosion casts

Studies of microvascular geometry made from microscope observations of tissues in vivo or after perfusion with a silastic elastomer or india ink are restricted to a two-dimensional field of view. Microvascular corrosion casts, however, if of sufficient rigidity and structural integrity, can yield three-dimensional information when examined under the scanning electron microscope. We have used modified Batsons No. 17 anatomical casting compound (having a shrinkage less than 1% on setting) to prepare casts of the microvasculature of the heart and skeletal muscles in anesthetized rats. In casts from the L. ventricle the capillary network appeared to parallel the arrangement of the muscle fibers, but showed many capillary loops and anastomoses. In skeletal muscles (gastrocnemius and gracilis) held at full extension, in situ, the casts showed long straight capillaries with fewer branchings than in the heart. In shortened skeletal muscle the capillaries exhibited an undulatory configuration. Capillary diameters (mean +/- SD) were 5.14 +/- 1.42 micrometers (N = 202), 5.04 +/- 1.45 micrometers (N = 294) and 4.84 +/- 1.97 micrometers (N = 335) in L. ventricle, gastrocnemius, and gracilis muscles (both shortened), respectively. The mean values for capillary diameter in these three tissues did not differ significantly. Combining our data with those of L. Henquell, P. L. LaCelle, and C. R. Honig on erythrocyte deformability in the rat (Microvasc. Res. 12, 259-274 (1976)) suggests that even when the capillary bed is fully distended the smallest capillaries, amounting to 1-2% of the total number, must be channels for plasma flow alone. In cross-sectional views of the casts from contracted skeletal muscle the capillaries appeared to form a tightly meshed network of convoluted vessels around the fibers, such that in some regions a large fraction of the surface of each fiber was in contact with blood. The Krogh cylinder geometry appears not to be appropriate for modeling O2 transport in maximally shortened skeletal muscle; a more appropriate model may be that of a cylindrical muscle fiber supplied, at any point down its length, by a uniform peripheral O2 supply.

The heme oxygenase system: its role in liver inflammation.

Heme Oxygenase is the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron and bilirubin. To date, three heme oxygenase isozymes have been identified: HO-1, HO-2 and HO-3. While HO-1 is structurally different from its counterparts, HO-2 and HO-3 are very similar (90% homology), with HO-3 being a poor heme catalyst. Of the three isozymes, HO-1 is believed to be the only inducible form. Constitutively expressed HO-2 has been identified in several organs including kidney and vascular smooth muscle, with the most abundant sources (and activity) being in the liver, brain, spleen and testes. Within the normal liver, HO-2 is constitutively expressed within hepatocytes, Kupffer cells, endothelial cells and Ito cells. Until recently, products of the HO reaction were regarded as potentially toxic waste destined only for excretion. However, this view is changing as evidence suggests that HO activity plays an important protective role against cellular stress during inflammatory diseases. Biliverdin is reduced to bilirubin, which has been shown to possess potent antioxidative properties. CO, which is produced in equimolar concentrations to biliverdin and ferrous iron during heme oxidation by HO, may function as a second messenger stimulating soluble guanylate cyclase (sGC) and regulating vascular tone in combination with the free radical gas NO. CO may also possess anti-inflammatory properties such as the capacity to inhibit platelet aggregation, or the expression of pro-inflammatory cytokines. Recently, it has been shown that CO regulates bile formation and bile flow. We review the functional role of HO in liver and the potential application of HO-1 in therapeutic approaches to the treatment of inflammation.

Inhalation of carbon monoxide prevents liver injury and inflammation following hind limb ischemia/reperfusion

The induction of heme oxygenase (HO), the rate limiting enzyme in the conversion of heme into carbon monoxide (CO) and biliverdin, limits liver injury following remote trauma such as hind limb ischemia/reperfusion (I/R). Using intravital video microscopy, we tested the hypothesis that inhaled CO (250 ppm) would mimic HO‐derived liver protection. Hind limb I/R significantly decreased sinusoidal diameter and volumetric flow, increased leukocyte accumulation within sinusoids, increased leukocyte rolling and adhesion within postsinusoidal venules, and significantly increased hepatocyte injury compared with naïve animals. Inhalation of CO alone did not alter any microcirculatory or inflammatory parameters. Inhalation of CO following I/R restored volumetric flow, decreased stationary leukocytes within sinusoids, decreased leukocyte rolling and adhesion within postsinusoidal venules, and significantly reduced hepatocellular injury following hind limb I/R. HO inhibition did not alter microcirculatory parameters in naïve mice, but did increase inflammation, as well as increase hepatocyte injury following hind limb I/R. Inhalation of CO during HO inhibition significantly reduced such microcirculatory deficits, hepatic inflammation, and injury in response to hind limb I/R. In conclusion, these results suggest that HO‐derived hepatic protection is mediated by CO, and inhalation of low concentrations of CO may represent a novel therapeutic approach to prevent remote organ injury during systemic inflammatory response syndrome, or SIRS.

CORM-3-derived CO modulates polymorphonuclear leukocyte migration across the vascular endothelium by reducing levels of cell surface-bound elastase.

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

Capillary configuration and fiber shortening in muscles of the rat hindlimb: Correlation between corrosion casts and stereological measurements

It has been a matter of discussion whether the dramatic increase in capillary tortuosity visualized in shortened muscles by vascular cast represents in vivo situations. In this study, we combined vascular cast and stereological methods in the same samples, in order to obtain (1) measurements of sarcomere length in the same muscles from which corrosion casts were prepared, and (2) scanning electron micrographs of the three-dimensional arrangement of capillaries in the same muscles where capillary anisotrophy was estimated by morphometry. Various rat skeletal muscles (soleus, gastrocnemius, and gracilis) were examined at lengths ranging from full shortening to full extension. We found a very good correlation between capillary geometry in material prepared for vascular casts and in muscles perfusion-fixed in situ. All muscles, cast and noncast, showed the same progressive curvilinear decrease in capillary anisotropy with decreasing sarcomere length. Capillary tortuosity visualized by corrosion casts in shortened muscles is a consequence of fiber shortening, within physiological sarcomere lengths; it does not represent an artifact related to the casting procedure.

Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice

The role of haem oxygenase (HO) in the hepatic accumulation of leukocytes in mice during the initiation of remote organ injury following normotensive limb ischaemia‐reperfusion (I‐R) was investigated. Remote organ injury was initiated by 1 h bilateral hindlimb ischaemia followed by either 1 or 1.5 h reperfusion (I‐R) in male C57BL/6 mice. Mice were randomly assigned to either sham (no I‐R, n= 4), I‐R (n= 4 for both time points), I‐R plus chromium mesoporphyrin (CrMP, n= 4) to inhibit HO or I‐R plus haemin (n= 4) to increase HO. Leukocyte accumulation and leukocyte‐endothelial interaction were directly measured using fluorescence intravital microscopy. Leukocytes were labelled via an injection of rhodamine 6G. In sinusoids the total number and the number of stationary leukocytes were assessed. In postsinusoidal venules the number of adherent and rolling leukocytes and the velocities of both red blood cells and leukocytes were measured. The total number of leukocytes increased in sinusoids of I‐R mice reaching a plateau within 1 h compared with sham animals, while the number of stationary leukocytes progressively increased over the entire study period. Stationary leukocytes in sinusoids increased after 1 and 1.5 h of I‐R following CrMP, while they were significantly reduced following haemin treatment compared to animals treated with I‐R only. In postsinusoidal venules a progressive increase in adherent leukocytes also occurred. As observed in sinusoids, CrMP significantly increased, while haemin significantly reduced leukocyte adhesion. The number of rolling leukocytes increased after CrMP in both I‐R groups (1 and 1.5 h). The velocities of rolling leukocytes declined following 1.5 h of I‐R compared with sham. Haemin treatment of 1.5 h I‐R animals restored the velocities back to sham levels. The calculated wall shear rates in postsinusoidal venules were significantly lower in all I‐R groups in comparison to sham animals. Combination of 1.5 h I‐R with CrMP resulted in the lowest shear rates of all I‐R groups. The number of stationary leukocytes within sinusoids and adherent leukocytes in postsinusoidal venules were correlated to the corresponding alanine aminotransferase (ALT) levels. In conclusion, endogenous HO reduces leukocyte‐endothelial interactions within the liver. Thus, endogenous HO activity provides an important mechanism controlling the hepatic inflammatory response during the initiation of remote organ injury following normotensive limb ischaemia‐reperfusion.

Cytokines contribute to early hepatic parenchymal injury and microvascular dysfunction after bilateral hindlimb ischemia.

PURPOSE Hepatic dysfunction may contribute to death from multiple organ dysfunction after abdominal aortic surgery. Several factors are likely responsible, and the purpose of this study was to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are involved in initiating this remote hepatic injury. METHODS In a normotensive rat model of 4-hour bilateral hindlimb ischemia/reperfusion (I/R), we measured systemic TNF-alpha and IL-1 levels throughout the I/R period. Rats were randomly assigned to either the 3-hour control group, the 3-hour I/R group, or the I/R group with administration of a polyclonal antibody (PAb) to TNF-alpha (I/R + TNF-alpha PAb). Direct evidence of lethal hepatocyte injury through the labeling of nuclei by propidium iodide (per 10(-1)mm(3)) and altered microvascular perfusion were assessed by using intravital microscopy. RESULTS Systemic TNF-alpha peaked at 83.97 pg/mL (P <.05, n = 5) at 30 minutes of reperfusion and returned to baseline in 60 to 90 minutes. No significant change in systemic IL-1 was detected (P <.05, n = 4). Alanine aminotransferase increased 2.5-fold in the I/R group through 3 hours of reperfusion (P <.05, n = 4), and TNF-alpha PAb did not attenuate this alanine aminotransferase increase (P <.05, n = 6). Lethal hepatocyte injury increased by 8-fold in the I/R group compared with the control group (P <.05, n = 5), whereas TNF-alpha PAb significantly reduced this injury (P <.05, n = 4). No regional differences in injury were noted within the acinus. Total perfusion within the microvascular unit did not drop; however, significant flow heterogeneity was observed. The proportion of continuously perfused sinusoids declined in the I/R group after 3 hours of reperfusion in both periportal (62.0 +/- 2.2, P <.05) and, to a lesser, although significant, degree, in the pericentral regions (73. 2 +/- 1.73, P <.05). CONCLUSION By scavenging extracellular TNF-alpha with a PAb, we provide direct evidence that TNF-alpha contributes to, but is not solely responsible for, early remote hepatocellular injury and microvascular dysfunction. The administration of TNF-alpha PAb reduced lethal hepatocyte injury in both regions of the acinus and also improved perfusion in the periportal region (76.8 +/- 5.41, P <.05), but not in the pericentral region. This suggests that TNF-alpha released during reperfusion mediates early remote hepatocellular injury and microvascular dysfunction after a remote ischemic insult.

Effect of sarcomere length on total capillary length in skeletal muscle: In vivo evidence for longitudinal stretching of capillaries

It is generally assumed that when a muscle is shortened or extended the total length of capillaries does not change, implying that capillaries are nondistensible, longitudinally. On the basis of stereological estimates of capillary anisotropy versus sarcomere length, we propose that as long as capillaries are in a tortuous configuration muscle extension will merely decrease the tortuosity, leaving vessel length unaltered. Once capillaries have been pulled into a straight configuration, further extension of the muscle will cause the vessels to stretch. By means of intravital videomicroscopy we have demonstrated that stretching of individual capillaries does indeed occur over a sarcomere length range of 2.1 to 2.9 microns in rat extensor digitorum longus muscle. In vivo measurements of the lengths of six capillaries together with the sarcomere lengths of adjacent fibers were made in muscles positioned at various degrees of extension. Normalized data indicated that four capillaries stretched to the same degree as the muscle, one stretched more and another less. This may reflect differences in distensibility or tortuosity of capillaries in series with one another. The elastic stretching of capillaries during muscle activity may have important consequences in terms of shifts in permeability and increases in capillary surface area.

Ischemic tolerance in skeletal muscle: role of nitric oxide.

We tested the hypothesis that ischemic preconditioning (PC) of skeletal muscle provided tolerance to a subsequent ischemic event 24 h later, and that such protection was due to nitric oxide (NO). Male Wistar rats, anesthetized with halothane, were randomly assigned to groups: ischemic (no PC; n = 11), PC ( n = 11), PC + N-nitro-l-arginine methyl ester (l-NAME; 100 μmol/l; n = 5), PC + N-nitro-d-arginine methyl ester (100 μmol/l; n= 4), PC + aminoguanidine (AMG; 100 μmol/l; n = 4), ischemic +l-NAME ( n= 4), or ischemic + AMG ( n = 4). PC consisted of 5× 10 min of ischemia and reperfusion, and, 24 h later, 2 h of ischemia were induced by a tourniquet applied to the limb. With the use of intravital microscopy, the number of perfused capillaries ( N pc) in the extensor digitorum longus (EDL) muscle was measured over a 90-min reperfusion period. The ratio of ethidium bromide- to bisbenzimide-labeled nuclei was used to estimate tissue injury. PC preserved N pc(23.6 ± 2.5) following 2 h of ischemia compared with sham muscles (11.5 ± 5.1), significantly elevating inducible NO synthase (iNOS) activity (81% increase), but did not afford protection to the parenchyma.l-NAME and AMG prevented ischemia-reperfusion-induced reduction in N pc in muscles without PC. However, after 90 min of reperfusion,l-NAME ( N pc = 15.0 ± 1.7), but not AMG ( N pc = 22.8 ± 3.1), significantly reduced the microvascular protection afforded by PC. We conclude that PC of the EDL muscle resulted, 24 h later, in protection to microvascular perfusion only, and that such protection was due to NO from sources other than iNOS.We tested the hypothesis that ischemic preconditioning (PC) of skeletal muscle provided tolerance to a subsequent ischemic event 24 h later, and that such protection was due to nitric oxide (NO). Male Wistar rats, anesthetized with halothane, were randomly assigned to groups: ischemic (no PC; n = 11), PC (n = 11), PC + N-nitro-L-arginine methyl ester (L-NAME; 100 micromol/l; n = 5), PC + N-nitro-D-arginine methyl ester (100 micromol/l; n= 4), PC + aminoguanidine (AMG; 100 micromol/l; n = 4), ischemic + L-NAME (n= 4), or ischemic + AMG (n = 4). PC consisted of 5x 10 min of ischemia and reperfusion, and, 24 h later, 2 h of ischemia were induced by a tourniquet applied to the limb. With the use of intravital microscopy, the number of perfused capillaries (Npc) in the extensor digitorum longus (EDL) muscle was measured over a 90-min reperfusion period. The ratio of ethidium bromide- to bisbenzimide-labeled nuclei was used to estimate tissue injury. PC preserved Npc (23.6 +/- 2.5) following 2 h of ischemia compared with sham muscles (11.5 +/- 5.1), significantly elevating inducible NO synthase (iNOS) activity (81% increase), but did not afford protection to the parenchyma. L-NAME and AMG prevented ischemia-reperfusion-induced reduction in Npc in muscles without PC. However, after 90 min of reperfusion, L-NAME (Npc = 15.0 +/- 1.7), but not AMG (Npc = 22.8 +/- 3.1), significantly reduced the microvascular protection afforded by PC. We conclude that PC of the EDL muscle resulted, 24 h later, in protection to microvascular perfusion only, and that such protection was due to NO from sources other than iNOS.

Intestinal mucosal permeability to 51Cr-ethylenediaminetetraacetic acid is increased after bilateral lower extremity ischemia-reperfusion in the rat*

BACKGROUND Despite successful revascularization of ischemic extremities, multiorgan dysfunction syndrome develops in some patients. Mechanisms responsible for this are not known; however, the gastrointestinal tract has been implicated as a possible mediator. Our objective was to demonstrate increased intestinal mucosal permeability after bilateral hindlimb ischemia-reperfusion (I-R) in a rodent model. METHODS Sixteen male Wistar rats were randomized either to 4 hours of bilateral hindlimb tourniquet ischemia and 24 hours of reperfusion (n = 8) or control groups (n = 8). The animals received 10 MuCi 51Cr-ethylenediaminetetraacetic acid (EDTA) by gavage, and excretion was measured in urine collected every 8 hours in 16 animals and every 4 hours in 8 animals. Arterial blood pressure was monitored continuously. Intravenous normal saline solution (3 ml/hr) with fentanyl (2 microgram/100 gm/hr) was continuously administered. Immediately before death complete blood count and levels of arterial lactate, creatinine, and urea were obtained. Mesenteric lymph nodes were harvested from the ileocecal region and cultured. Distal small bowel and proximal colon were preserved for histologic analysis. An additional 11 rats, six experimental and five control, were evaluated for mesenteric lymph node cultures only. RESULTS Urinary excretion of 51Cr-EDTA was significantly greater in the I-R group between 0 and 8 hours (p < 0.02) and 8 to 16 hours (p < 0.0002) of reperfusion. This increase occurred as early as 4 to 8 hours of reperfusion (p < 0.0001). Urine volume in the I-R group was significantly reduced during 0 to 4 hours of reperfusion (p < 0.002). Hemoglobin and lactate level were significantly different in the I-R group. Leukocyte and platelet counts, levels of creatinine and urea, and colony counts from mesenteric lymph nodes were similar in I-R and control groups. Blinded histologic analysis of bowel segments did not reveal morphologic differences. CONCLUSIONS Bilateral hindlimb I-R produces remote intestinal mucosal injury shown by significantly increased permeability to 51Cr-EDTA. Such increased mucosal permeability may be important in the development of multiorgan dysfunction syndrome in patients who sustain lower extremity I-R injury.