Local heat preconditioning of myocutaneous tissue markedly increases flap survival by maintaining adequate nutritive perfusion rather than inducing ischemic tolerance. The protection is caused by the increased arteriolar blood flow due to significant arteriolar dilation, which is mediated through the carbon monoxide-associated vasoactive properties of HSP-32.
In the present study in a murine model of chronic ischaemia, we analysed: (i) whether aging was associated with an increased susceptibility to ischaemic necrosis, and (ii) whether this was based on microvascular dysfunction or reduced ischaemic tolerance. An ischaemic pedicled skin flap was created in the ear of homozygous hairless mice. The animals were assigned to three age groups, including adolescent (2±1 months), adult (10±2 months) and senescent (19±3 months). Microvascular perfusion of the ischaemic flap was assessed over 5 days by intravital microscopy, evaluating FCD (functional capillary density), capillary dilation response and the area of tissue necrosis. Expression of the stress-protein HO (haem oxygenase)-1 was determined by immunohistochemistry and Western blotting. Induction of chronic ischaemia stimulated a significant expression of HO-1 without a significant difference between the three age groups. This was associated with capillary dilation, which, however, was more pronounced in adolescent (10.5±2.8 μm compared with 3.95±0.79 μm at baseline) and adult (12.1±3.1 μm compared with 3.36±0.45 μm at baseline) animals compared with senescent animals (8.5±1.7 μm compared with 3.28±0.69 μm at baseline; P value not significant). In senescent animals, flap creation further resulted in complete cessation of capillary flow in the distal area of the flap (FCD, 0±0 cm/cm2), whereas adult (11.9±13.5 cm/cm2) and, in particular, adolescent animals (58.4±33.6 cm/cm2; P<0.05) were capable of maintaining residual capillary perfusion. The age-associated microcirculatory dysfunction resulted in a significantly increased flap necrosis of 49±8% (P<0.05) and 42±8% (P<0.05) in senescent and adult animals respectively, compared with 31±6% in adolescent mice. Of interest, functional inhibition of HO-1 by SnPP-IX (tin protoporphyrin-IX) in adolescent mice abrogated capillary dilation, decreased functional capillary density and aggravated tissue necrosis comparably with that observed in senescent mice. Thus aging is associated with an increased susceptibility to tissue necrosis, which is due to a loss of vascular reactivity to endogenous HO-1 expression, rather than a reduction in ischaemic tolerance.
Using intravital microscopy in a chronic in vivo mouse model, we studied the demarcation of myocutaneous flaps and evaluated microvascular determinants for tissue survival and necrosis. Chronic ischemia resulted in a transition zone, characterized by a red fringe and a distally adjacent white falx, which defined the demarcation by dividing the proximally normal from the distally necrotic tissue. Tissue survival in the red zone was determined by hyperemia, as indicated by recovery of the transiently reduced functional capillary density, and capillary remodeling, including dilation, hyperperfusion, and increased tortuosity. Angiogenesis and neovascularization were not observed over the 10-day observation period. The white rim distal to the red zone, appearing as "falx lunatica," showed a progressive decrease of functional capillary density similar to that of the necrotic distal area but without desiccation, and thus transparency, of the tissue. Development of the distinct zones of the critically ischemic tissue could be predicted by partial tissue oxygen tension (Pt(O(2))) analysis by the time of flap elevation. The falx lunatica evolved at a Pt(O(2)) between 6.2 +/- 1.3 and 3.8 +/- 0.7 mmHg, whereas tissue necrosis developed at <3.8 +/- 0.7 mmHg. Histological analysis within the falx lunatica revealed interstitial edema formation and muscle fiber nuclear rarefaction but an absence of necrosis. We have thus demonstrated that ischemia-induced necrosis does not demarcate sharply from normal tissue but develops beside a fringe of tissue with capillary remodeling an adjacent falx lunatica that survives despite nutritive capillary perfusion failure, probably by direct oxygen diffusion.
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