Immunology & Immunological Disorders Poster Session
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MR And DBT.
The ability of animals to maintain their DBT in response to the cold challenge was noted. All young animals were able to maintain their DBT above 18 °C until the end of the 2-2.5 hour cold challenge. Some of the old animals were able to maintain their DBT above 18 °C while others were unable to do so. On the basis of these findings obtained from young animals, the old animals were sub classified among those that were able to maintain their DBT above 18 °C and those that were unable to maintain DBT to this level. This cut-off temperature of 18 °C was arbitrary in nature and is not a physiological cut-off point. The statistical analysis was done using two sample student’s t-Test .
Table 1: DBT and MR of Mice exposed to a cold challenge.
First 3 Days 1 hour on Test Day -------------------------------------------------------------------- DBT (Young) 36.8 (0.21) 37.6 (0.39) MR (Young) 70.8 (4.68) 74.3 (12.94) DBT (Old, above18 °C) 36.5 (0.14) 37.6 (0.54) MR (Old, above 18 °C) 56.8 (1.84) 68.5 (10.38) DBT (Old, below18 °C) 36.7 (0.13) 36.8 (0.26) MR (Old, below18 °C) 59.4 (3.44) 59.2 (9.21)
In Tables 1, mean DBT and MR recordings of mice on the first 3 days of the experiment are compared with one hour [9:30-10:30] recording on the test day. The values shown are means (±SEM) of five observations, in °Celsius for DBT and ml O2/kg/min for MR. It is evident by the data recorded on first three days that the basal metabolic rate (BMR) of young mice was significantly higher than the old mice (p < 0.05, two-tail analysis and p < 0.02, one-tail analysis). There was no statistically significant difference between the two subgroups of old mice (p > 0.05; both on two-tail and one-tail analysis).
Mean DBT And MR Of Mice During The Cold Challenge.
Figure 1 shows the mean (±SEM) of DBT and MR recordings of young animals during the cold challenge. The temperature drop started at time “zero” on the ‘x’ axis; the initial temperature was 20 °C. The drop rate was maintained around 1 °C every three minutes. The arrow in Figure 1 indicates the time at which the final lowest temperature of 4 °C was achieved. There was a mean decrease in DBT and MR of 19% and 13%, respectively.
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Figure 1: Mean Value For MR And DBT Of Young Mice In A Cold Challenge.
Figure 2 shows the mean values (±SEM) of DBT and MR for those old mice which were able to maintain their DBT above 18 °C in the cold challenge. The mean DBT gradually declined from around 37 °C at the beginning of the cold challenge to about 31 °C at the end of the experiment. The fall of DBT was about 16%. Mean MR, like in the young animals, showed an increase in the early part of the cold challenge but later decreased to about 67 ml O2/kg/min. A mean decrease of about 16%.
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Figure 2: Mean Value For MR And DBT Of Old Mice (Which Were Able To Maintain DBT Above 18 °C) In A Cold Challenge.
Figure 3 shows the mean values (±SEM) of DBT and MR for those old mice which were unable to maintain their DBT above 18 °C in a cold challenge. A difference in the pattern was observed in these old mice. Mean DBT at the start of cold challenge was around 37 °C. This temperature was maintained only for the initial 20 minutes of the cold challenge. Thereafter a decline in DBT was evident for the next 60 minute, until the mean value reached 30 °C. Then the slope of the decline increased sharply and the mean DBT at the end of the experiment was about 12 °C. The mean DBT declined from around 37 °C at the beginning of the cold challenge to about 12 °C at the end of the experiment. The fall of DBT was 68%.
Although mean MR showed an increase in the early part of the cold challenge, it was unable to maintain itself to what it was at the beginning of the cold challenge. Mean MR decreased from 75 ml O2/kg/min at the beginning of cold challenge to 15 ml O2/kg/min at the end. The decrease in mean MR was 80%.
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Figure 3: Mean Value For MR And DBT Of Old Mice (Which Were Unable To Maintain DBT Above 18 °C) In A Cold Challenge.
Table 2: Effect Of The Cold Challenge On IL-1 Binding In Young And Old Mice.
Binding in Hypothalamus
cold challenge No cold challenge
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+ IL1 - IL1 + IL1 - IL1
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Young 305.03 475.84 51.91 40.90
(166.94) (323.52) (7.53) (6.46)
Old (above 18 °C) 326.60 280 (129.05) (136.75)
Old (below 18 °C) 449.05 649.18 (262.49) (442.28
Old 167.90 81.66 (72.28) (25.65)
Comparison of interleukin-1ß binding in hypothalamus between young and old mice exposed to a cold challenge against the group of animals not exposed to the cold challenge. The values are expressed in fmoles/mg of protein and depict means (±SEM). The data show increased binding among both young and old animals both in the presence (+IL1) and absence (-IL1) of unlabelled IL-1; but IL-1 receptor binding when exposed to a cold challenge showed a varied response and thus statistical significance was not achieved (p > 0.05).
Table 3: Effect Of The Cold Challenge On IL-1 Binding In Young And Old Mice.
Binding in Hippocampus
Cold challenge No cold challenge
-----------------------------------------------------------------------
+ IL1 - IL1 + IL1 - IL1
-----------------------------------------------------------------------
Young 100.93 123.44 71.81 57.02
(28.31) (44.57) (6.80) (13.08)
Old (above 18 °C) 435.70 312.99 (327.88) (195.37)
Old (below 18 °C) 614.38 909.04 (272.05) (489.52)
Old 127.20 107.42 (63.84) (50.88)
Comparison of interleukin-1ß binding in hippocampus between young and old mice exposed to a cold challenge against the group of animals not exposed to the cold challenge. The values are expressed in fmoles/mg of protein and depict means (±SEM). The data show increased binding in those old animals which were able to maintain DBT above 18 °C; both in the presence (+IL1) and absence (-IL1) of unlabelled IL-1. It is also evident that the old animals exposed to cold showed larger ±SEM.
When the data was combined to investigate the effect of cold challenge, it was expected that the animals exposed to the cold challenge should show an increased binding compared to controls. This effect was expected because of the known role of IL-1 in thermogenesis. On the basis of this one-tail analysis was deemed appropriate. An increased binding was evident in both hippocampus and hypothalamus in the presence (+IL1) of interleukin-1 (p < 0.05). An increased binding was also observed in hypothalamus (p < 0.05) and hippocampus (p < 0.025) in the absence (-IL1) of interleukin-1 (Table 4).
Table 4: Effect Of The Cold Challenge On IL-1 Binding [Combined Data].
------------------------------------------------------------------
Cold challenge No cold challenge
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Hypothalamus (+ IL1) 381.1 (138.3) 109.9 (38.16)
Hippocampus (+IL1) 746.85 (355.6) 99.5 (31.82)
Hypothalamus (- IL1) 525.78 (236.95) 61.28 (13.81)
Hippocampus (- IL1) 772.21 (331.95) 82.22 (26.1)
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Binding data obtained in the presence and abscence of unlabelled IL-1 was pooled from old and young mice to give the values shown. All values are expressed in fmoles/mg of protein. The data show increased binding of [125I IL-1ß] in hypothalamus and hippocampus when exposed to the cold challenge in the presence of unlabelled interleukin-1 (+ IL1). In both instances the increase was statistically significant (p < 0.05). When analysed in the absence of unlabelled interleukin-1 (- IL1), the data reached statistical significance for both hippocampal (p < 0.025) and hypothalamic binding (p < 0.05).
Immunohistochemistry.
ABC Vectastain® Kit was used for immunohistochemistry performed on 10 microns thick sections of hippocampus and hypothalamus. The figures to follow show increased expression of IL-1 in old animals compared to the young animals and in the animals exposed to a cold challenge compared to the ones not facing a cold challenge. This is most evident in those old animals that were unable to maintain their DBT above 18 °C upon exposure to a 4 °C temperature drop. IL-1 expression is evident in hypothalamus, especially in those old animals which were unable to maintain their DBT upon exposure to the cold challenge. IL-1 expression is also evident in the pyramidal cells of CA3 region and dentate gyrus (DG) of hippocampus.
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Figure 4: Hypothalamus, Control Tissue (X40).
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Figure 5: Hippocampus, Control Tissue (X10).
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Figure 6: 2 Month Old Animal; Hypothalamus (X40).
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Figure 7: 12 Month Old Animal; Hypothalamus (X40).
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Figure 8: 2 Month Old Animal; Hippocampus (X4).
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Figure 9: 12 Month Old Animal; Hippocampus (X4).
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Figure 10: 2 Month Old Animal; Hypothalamus (X40; Cold Challenge).
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Figure 11: 12 Month Old Animal; Hypothalamus (X40; Able To Maintain DBT In Cold Challenge).
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Figure 12: 12 Month Old Animal; Hypothalamus (X40; Unable To Maintain DBT In Cold Challenge).
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Figure 13: 2 Month Old Animal; Hippocampus (X4; Cold Challenge).
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Figure 14: 12 Month Old Animal; Hippocampus (X4; Able To Maintain DBT In Cold Challenge).
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Figure 15: 12 Month Old Animal; Hippocampus (X4; Unable To Maintain DBT In Cold Challenge).
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