Measuring Skin Blood Flow and Vascular Permeability
Published: Last Edited:
Disclaimer: This essay has been submitted by a student. This is not an example of the work written by our professional essay writers. You can view samples of our professional work here.
Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.
The aim of this experiment is to compare the dose-related inflammatory response demonstrated by the skin following injection of histamine and bradykinin - two inflammatory mediators. Methods used will demonstrate a non-invasive, quantitative way to measure blood flow and vascular permeability in the skin.
The acute inflammatory reaction occurs to protect the body in response to a pathogen or other noxious substance. There are two components: adaptive immunological response (which is described as a more specific immune response) and the innate response which occurs immediately upon infection and consists of both vascular and cellular effects (Rang and Dale, 2007). The innate response will be studied in this experiment, specifically in the skin.
Bradykinin and histamine are inflammatory mediators involved in the innate response and will be studied at different doses. The results can be used to provide a potential target for therapeutic use: further experimentation would allow the addition of inflammatory mediator antagonists to potentially reduce the four cardinal signs of inflammation: pain, heat, redness and swelling.
The local oedema and vasodilation give rise to the 'wheal and flare.' The reddening represents vasodilation of small arterioles, and the increased permeability of the post capillary venules is represented by the 'wheal'. The 'flare' occurs due to stimulation of sensory nerves causing release of vasodilators. This is known as the triple response. It will be the 'wheal and flare' that will be measured and used to describe the action of the two inflammatory mediators.
The methods used were in-vivo; the doses of inflammatory mediators (and saline control) were injected into 10 volunteers. It was confirmed there were no known allergies to either bradykinin or histamine and all correct health and safety procedures were followed.
Each of the 10 subjects were injected first with 25μL of saline solution, used as a control to show there was nothing in the saline (that the inflammatory mediators were diluted with) causing an inflammatory response. This was followed by doses of 10, 30, and 100μM histamine for 5 subjects and the same doses of bradykinin for the other 5 was added, all at 30 second intervals. These were administered using a sterilised syringe which contained the correct concentration. The doses were injected into forearm intradermally and care was taken to ensure the complete volume of 25μL was taken up by the skin. Each successive administration was slightly further up the arm giving space for each of the four doses and to try and prevent the 'flares' from overlapping.
At periods of 2, 5, 10 and 15mins a clear sheet of acetate was placed over the centre of injection and the 'wheal' and 'flare' were circled using a non-wipe pen and repeated
for each respective dose. This provided the area of the 'wheal' and 'flare' at each of the given concentrations at each of the given periods of time following injection, for each respective inflammatory mediator. The 'flare' was cut from the acetate and weighed accurately to 4 decimal places. Subsequently, the 'wheal' was cut from the centre of the 'flare' and was also weighed. This process was repeated for each of the doses of inflammatory mediator (bradykinin and histamine) and for the saline control also. A 2cm2 square was ruled onto the acetate which was also cut out and weighed. This provided a conversion between weight and area, allowing the area of the 'wheal and flares' to be calculated (credit to Dr. Dean Willis).This data was tabulated and can be found in the appendix and illustrated in the results.
The data was checked for any anomalous values that could be defined as incorrect based upon logical criteria. 'Group 1' for the histamine set had flare sizes of 0cm2 however, had wheal sizes greater than this. Therefore this data was removed to all analysis as it is clearly incorrect.
The data was then averaged for each of the 5 subjects for both histamine and bradykinin. There were two independent variables: time and concentration; and two dependant variables: wheal and flare areas. The independent variables were illustrated on separate graphs and the wheal and flare sizes were imposed on the same.
To produce graphs to illustrate the change in area with concentration, first the largest average value recorded for each concentration was selected and tabulated. This allows comparison not only between different concentrations of the same mediator, but also between bradykinin and histamine. This also means time was irrelevant because it did not matter at which time recording the values were selected The increase in wheal or flare size due to inflammatory mediator was calculated (i.e. the difference between the wheal or flare recorded and saline). This 'increase' in wheal or flare was plotted against the respective concentration and the concentration was plotted in log scale to illustrate a dose-response curve.
To illustrate the change in area with respect to time, firstly, the data was scanned to select a concentration at which the change in wheal and flare was best illustrated. This concentration was taken to be 100μM (for both mediators to ensure continuity and to allow comparison). The Average wheal and flare size was then plotted against time for both bradykinin and histamine.
Removed data: (see appendix) Group 1 of the histamine section has a flare size of 0.000 recorded with a wheel size of greater than this. This is likely to be a systematic error in not realising the flare is indeed underneath the wheel and not visible, in this case the flare is the same area of the wheel. However this is just speculation, and in order to ensure all data used is correct saline recordings for each time interval - both wheel and flare areas for group 1(histamine) were removed from analysis.
The wheal size only increased slowly with increased concentration of bradykinin to a maximum of 0.414 at 100μM. The value at 10μM was actually lower than that for saline. This is not a significant decrease however as it was taken as a decrease of 0.04cm2, which is a small area and the limitations of the experiment are likely to be the cause. The flare size, however, increased more with increasing concentration. The size of the flare is likely to represent a dose-response curve with a classic sigmoid shape if the concentration of bradykinin were to be increased further. However, due to the nature of the experiment this would not be practical as a much large concentration of inflammatory mediator could be dangerous for the subject.
It is also shown that the maximum flare area at 100μM was recorded at 10mins. It can therefore be deduced that it was relatively slow acting; however it cannot be determined whether the maximum value was indeed at 10mins - recorded as 7.808cm2. Equally the flare area could have rose to a maximum between 5-10mins and decreased, or rose to a maximum after 10mins and reduced to that recorded at 15mins.
It can be shown that at the lowest concentration (10μM) of histamine that there is only a small difference of 1.194cm2 between the maximum flare-area recorded by bradykinin. It can therefore be deduced that histamine caused a larger flare than bradykinin at the same concentrations. Ahe general trend is similar to that of bradykinin: small increase in wheal area, large increase in flare area. The maximum wheal area was only 0.03m2 larger than that recorded by bradykinin.
Again, the wheal area had very little variation with time: increase of 0.2cm2. The flare area was at a maximum recording of 18.625cm2 after just 2mins. Therefore, it is likely to have been at the maximum area before 2mins. This shows that histamine is faster acting than the bradykinin. There is a relatively linear decrease with time to a minimum value of 9.120cm2 recorded at 15mins. The flare area did of course continue to decrease after the 15minute period until there was no apparent inflammation, likewise for bradykinin.
As mentioned previously, the innate inflammatory response consists of both vascular and cellular effects. Vascular events begin by dilation of post capillary venules, causing an increased blood flow. Vasodilation is caused by the action of histamine (and other inflammatory mediators), leading to increased local blood flow and an increased vascular permeability - causing a local oedema. The fluid contains the components a proteolytic enzyme cascades producing bradykinin. Bradykinin is also an inflammatory mediator causing further vasodilation and vascular permeability leading to local redness and oedema respectively. This gives rise to the cardinal signs of inflammation: redness, swelling, heat and pain (also loss of function). The sensation of heat and pain ascend through sensory neurones via the spinothalamic tract.
Upon the presence of a pathogen, pathogen associated molecular patterns (PAMPs) are recognised on the surface of bacteria and causing the release of cytokines from macrophages. Cytokines are small polypeptides involved in cell-signalling and orchestrate inflammation. This allows expression of adhesion molecules in the endothelial cells. Phagocytes then adhere to the endothelium and migrate towards the bacteria where phagocytosis takes place. In addition, exudation of fluid occurs in response to an increased vascular permeability due to a combination of cytokine and inflammatory mediator action (as well as increased vasodilation in response to inflammatory mediators). The fluid allows four enzyme cascades to occur producing inflammatory further inflammatory mediators by proteolytic cleavage from their native (inactive) state. One of these cascades gives rise to bradykinin (Pocock and Richards, 2006).
Histamine is released in response to products of other enzyme cascade pathways such as C3a and c5a which make up part of the complement system. C3a and C5a bind with receptors on the surface of mast cells, causing a rise in intracellular calcium leading to exocytosis of histamine. Simple injection of bradykinin or histamine mimics these pathways.
Bradykinin is a vasodilator and also increases vascular permeability leading to a local swelling. This is consistent with the findings in this experiment. After Intradermal injection of bradykinin, the typical 'triple-response' was apparent; there was a wheel and flare as described by Sir Thomas Lewis. Breakdown is by kininases and it is likely to have cleaved bradykinin at a relatively fast rate due to the short lasting effect at 100μM where the flare area began to decrease after just 10mins.
Histamine has a similar action to bradykinin but found to act faster and also found to be more potent at each concentration tested. The flare area was at a maximum after just 2 minutes. Histamine acts on H1 receptors to dilate blood vessels, therefore it is likely there is a high expression of H1 receptors at the skin surface, or histamine has a great affinity for its receptor. It is likely to be a combination of both, however to confirm these ideas, experiments could be conducted on other tissue perhaps on organ tissue in-vitro using an animal model. This response is characteristic of the acute inflammatory pathway; however, more recent studies suggest that histamine has a role in chronic inflammation involved in the immune response (Jutel et al., 2009). There is regulation of T-cells (which make up part of the immune response) by H1 and H2 receptors. There is a 4th histamine receptor, H4 and further evidence for the role of histamine in chronic pathways comes from expression of H4 receptors on immune cells (Jutel et al., 2009).
It is apparent from figures 1 and 3 that an increase in either inflammatory mediator resulted in an increase in wheel area. As previously described, this is due to release of vasodilators from sensory nerves in response to stimulation. So it can be deduced that a larger concentration of bradykinin or histamine indicates a larger 'infection' and therefore the cascade process is accentuated. The wheal area stays relatively constant in both cases, this could be due to no addition action of inflammatory mediators on the vascular permeability, or indeed there is already a full effect - i.e. the post capillary venules are a permeable as possible. However another hypothesis could be that additional permeability would only lead to a further decreased extracellular solute concentration which would simply be reabsorbed by osmosis.
STRENGTHS AND LIMITATIONS
Strengths of the experiment were in that humans were used and methods were in-vivo. Therefore there is no reliance on animal models to use as a comparison. All subjects were of a similar age and gender was at random, hence, generally similar responses were found between each group. Limitations were found to be in injecting the inflammatory mediator intradermally. There was a tendency for not all of the solution to actually enter the skin, thus decreasing the number of moles of inflammatory mediator. This however did not seem to effect the results too greatly as 5 repeat groups would allow for some small error. It is still clear from the experiment that the aims were met and the mediators compared. Furthermore, measurement of the area was not particularly accurate. Firstly it was hard to judge the size of the wheel and flare and there was a tendency for the flares to overlap and was often left down to judgment of where to define the boundary. There were a few further cases where the wheel size exceed that of the flare (in addition the case described in the results) however these were only small differences and could easily have been to variations in the measurement of the weight. If the wheal and flare were the same size, the acetate could have been weighed twice and hence the small difference. This would not have affected the outcome of the experiment however so the data was accepted. Better methods of measurement of wheel and flare area would be to use an imaging technique and record the change in areas digitally. This would allow for calculation of the change in rate of area with respect to time (via differential equations) which would give a good indication as to the potency and allow for a more in-depth comparison.
Cite This Essay
To export a reference to this article please select a referencing stye below: