In this study we used real-time imaging technologies to better understand vascular changes associated mast cell degranulation within tissues. We found that the vascular response associated with mast cell degranulation is characterized by increased blood flow, thermal warming, vasodilation ( and S2
), and recruitment of collateral circulation (). Treatment with antihistamine partially restores the normal pattern of superficial blood flow and is associated with less tissue edema ( and S4
). To associate these changes with mast cell activation, we documented a rise in serum histamine following CHI in affected subjects () 
Histamine levels were elevated accordingly when patients were stratified based on severity (1). Serum tryptase levels remained unchanged () although local release of tryptase by mast cells was documented (). While it is known that tryptase is released by human mast cells together with histamine in vitro 
, there are discrepancies between the detection of these mediators in clinical disease 
attributed to the fact that mature tryptase occurs in complex with proteoglycan, which limits diffusion into the vascular compartment while it can be seen locally within the dermis associated with mast cells 
. The lack of detection of serum tryptase despite a rise in histamine entertains the possible contribution of histamine from basophils. This is unlikely for the following reasons: First, tryptase is also found in human basophils although the amount may vary considerably 
. Thus the absence of tryptase in blood could also be used as an argument against the involvement of blood born basophils in the cold induced reaction. Second, Kaplan et al., reported in an in vitro model of acquired cold urticaria that histamine was only induced in chilled skin tissues and not released from chilled blood leukocytes and purified basophils 
. Third, the serum of patients with cold urticaria does not activate basophils 
. Thus, these observations implicate mast cells as the primary source for the histamine-dependent reaction in patients with CUrt.
LSCI, IR and PLC simultaneously acquired images during CHI challenge. A pictorial comparison of the three modalities at baseline and 10 minutes post-CHI () displays a clear difference in dermal blood flow patterns and inflammatory response in patients versus controls. In patients, the marked inflammatory response is seen earlier in the hand than in the fingers (compare , to Figure S4B
). We were able to characterize the onset, peak and rewarming of tissue in patients and to identify hemodynamic markers. Using LSCI post-CHI, patients displayed significantly higher maximal, more delayed onset and slower recovery of mean blood flow (). IR imaging detected a rapid onset and exaggerated temperature response above baseline (). Color indexes using PLC revealed differences in delayed time to reach maximal color and recovery (). These findings support the conclusion that patients with CUrt during cold exposure have an exaggerated response to natural body heat preservation.
We demonstrated in a subset of individuals that the warming pattern of the hand and fingers is homogeneous in comparison to a patient with CUrt where there is a more rapid and dramatic recovery of the palm versus the fingers (). Given mast cell density is similar in superficial and deep dermis 
, the cooling of more tissue in the palm may lead to greater efflux of histamine and other vasodilator mediators and thus greater blood flow. At the other end of the spectrum, although the rewarming pattern following cold exposure in those with Raynaud’s syndrome is homogenous, it is much slower than controls due to dermal episodic ischemia 
. Our observations provide experimental support to those patients reporting variable regional responses to cold exposure.
When correlating microvascular changes to histamine, we demonstrated that the highest rate of change in response correlates with the rise in histamine ( and Video S1
). Repeat analysis of three patients on cetirizine highlights the utility of imaging to quantify a decreased superficial inflammatory response to cold challenge following administration of an anti-allergic drug ( and S4
). The spectrum of responses is likely a consequence of the need to block other mast cell derived or induced mediators in patients that have more severe pathology.
Using LSCI which assesses superficial blood flow, IR which reflects changes in blood flow in superficial and subcutaneous tissues and PLC which detects the skin reddening caused by both moving and stagnant red cells in blood capillary loops close to dermal papillae, we were able to examine the sequence of events in a local inflammatory response that occurs at different vascular plexuses within the skin microvascular bed. We thus observed that the time of onset, max rate of change, and mean time to reach maximum for three imaging signals did not dissociate (). This observation supports the conclusion that rewarming is associated with vasodilator recruitment of microvasculature in all vascular plexuses simultaneously. These vascular changes would be expected to facilitate the inflammatory response by increasing exposure of blood born cells and proinflammatory proteins to the area of insult.
Inducible inflammation in cold urticaria is a unique model that allows tissue mast cell dependent events to be studied in humans at baseline and during challenge testing. We were able to thus make a number of new and novel observations, which included the dermal phenotype of microvascular hypereactivity, time to recovery and response to antihistamine. It is tempting to speculate that patients with cold-induced urticaria are exhibiting an exaggerated protective response to cold whereby mast cell degranulation represents a protective mechanism to maintain blood flow and thus prevent more extensive tissue damage, much as has been suggested for cutaneous vasodilatation provoked by pressure