Cold Injury of the Rat Skin: A Fluorescence Histochemical Study of Adrenergic Nerves, Mast Cells and Patency of Cutaneous Blood Vessels

In order to investigate the pathogenesis of frostbite a combined trypan blue and catecholamine fluorescence method was adopted for studying changes in cutaneous adrenergic nerves, mast cells, the patency of blood vessels, the distribution of blood flow and vascular permeability after experimental cold injury. The skin of rats was frozen with a copper cylinder precooled in liquid nitrogen. The rats were investigated 1/2, 4, 8, 16 and 24 h or 2, 4, 7, 9 and 14 days after freezing. Trypan blue has a red fluorescence when absorbed onto proteins. In normal skin this stain was seen inside the blood vessels and in the dermal tissues. Bluegreen fluorescent adrenergic nerves were seen in the erector pili muscles and around arteries and arterioles. The fluorescence of the mast cells was yellow. In the specimens taken half an hour after skin freezing most of the adrenergic nerves appeared morphologically normal. Degenerative changes were observed in the mast cells varying from degranulation and diminution of yellow fluorescence to total disappearance of mast cell fluorescence. Increased extravasation of red trypan blue fluorescence was observed surrounding an area of diminished perfusion and poor trypan blue fluorescence. In the specimens taken 4, 8 and 16 h after freezing the central ischaemic area gradually grew and the trypan blue fluorescence in this area further diminished. This was surrounded by an area of increased trypan blue fluorescence and extravasation. A gradually increasing amount of degenerative change was seen in the adrenergic nerves especially 16 h after freezing. An accumulation of noradrenaline in the adrenergic nerves was observed at the margin of the frozen area and began allready 1 h after freezing, while the catecholamine fluorescence had totally disappeared from the nerves of the central ischaemic area 24 h after freezing. Neither was mast cell fluorescence seen in this area. In the early hours after freezing extravasation of red blood cells, erythrostasis and the development of aggregations of red blood cells was apparent. In addition total cessation of blood flow and thrombosis together with necrotic changes in the blood vessel wall in some blood vessels became apparent in the specimens taken 24 h after skin freezing. The first signs of the regeneration of adrenergic nerves from the margins towards the central area of injury were observed 4 days after freezing. It is suggested that the vasoconstriction produced by the action of adrenergic nerves plays an important role in the development of frostbite. After skin freezing noradrenaline liberated from the degenerating nerves, as well as catecholamine accumulation in the adrenergic nerves at the margin of the frozen area might further increase vasospasm. The increase in vascular permeability is probably partly due to vasoactive substances liberated from the mast cells after freezing injury.