Acute changes of pro-inflammatory markers and corticosterone in experimental subarachnoid haemorrhage: A prerequisite for severity assessment

Abstract

Many details of the pathophysiology of subarachnoid haemorrhage (SAH) still remain unknown, making animal experiments an indispensable tool for assessment of diagnostics and therapy. For animal protection and project authorization, one needs objective measures to evaluate the severity and burden in each model. Corticosterone is described as a sensitive stress parameter reflecting the acute burden, and inflammatory markers can be used for assessment of the extent of the brain lesion. However, the brain lesion itself may activate the hypothalamic-pituitary-adrenal-axis early after SAH, as shown for ischemic stroke, probably interfering with early inflammatory processes, thus complicating the assessment of severity and burden on the basis of corticosterone and inflammation. To assess the suitability of these markers in SAH, we evaluated the courses of corticosterone, IL-6 and TNF-α up to 6h in an acute model simulating SAH in continuously anaesthetized rats, lacking the pain and stress induced impact on these parameters. Animals were randomly allocated to sham or SAH. SAH was induced by cisterna magna blood-injection, and intracranial pressure and cerebral blood flow were measured under continuous isoflurane/fentanyl anaesthesia. Withdrawn at predetermined time points, blood was analysed by commercial ELISA kits. After 6h the brain was removed for western blot analysis of IL-6 and TNF-α. Serum corticosterone levels were low with no significant difference between sham and SAH. No activation of the HPA-axis was detectable, rendering corticosterone a potentially useful parameter for stress assessment in future chronic studies. Blood IL-6 and TNF-α increased in both groups over time, with IL-6 increasing significantly more in SAH compared to sham towards the end of the observation period. In the basal cortex, IL-6 and TNF-α increased only in SAH. The pro-inflammatory response seems to start locally in the brain, reflected by an increase in peripheral blood. An additional surgery-induced systemic inflammatory response should be considered.

Fig 1. Illustration of the experimental setup.

The positions of the cranial window for laser speckle contrast imaging, the silver wire for EEG recording and the catheters for blood injection and ICP assessment are shown.

Fig 2. Experimental design.

A: Diagram of sample size distribution: 32 animals were randomly allocated to four different groups for blood sampling for inflammation markers (IL-6 and TNF-α) and corticosterone; after subtracting drop-outs from surgery and recording phase, a total of 21 animals were included in the final analysis. Subarachnoid hemorrhage (SAH) was induced by injection of 500μl arterial blood within 1 minute into the cisterna magna, sham animals did not receive any injection. B: Time points for blood sampling for inflammatory cytokine and corticosterone analysis: The diagram shows the time points (x) when blood was withdrawn within basic surgery (anaesthesia, tracheotomy, ventilation, A./V. femoralis cannulation), head fixation and cranial preparation (turning to prone position, fixation with ear bars in stereotactic frame, cranial window preparation, trepanations for EEG and ICP recording, cannula for icv blood injection) followed by the measurement period; red flash marks the time point of SAH induction.

Fig 3. Box plots of IL-6 and TNF-α in blood samples from SAH (black) and sham (grey) animals.

Data are presented as boxplot diagrams; the whiskers represent the minimum and maximum values, the dots show single points. Data were analyzed using repeated measures 2-way ANOVA followed by multiple comparisons tests: Sidak´s (comparisons between the groups; (A, B) IL-6 (A) and TNF-α (B) sham: n = 6; SAH: n = 5) or Tukey´s ((C, D) for IL-6, comparisons within the group; only significances in respect to baseline are depicted); * p < 0.05; ** p < 0.01; dotted line: minimum detectable dose (IL-6: 21pg/ml, TNF-α 5pg/ml).

Fig 4

Box plots of corticosterone in blood samples from SAH (black) and sham (grey) Corticosterone was analysed at predefined sampling time points within the surgery and during the recording phase; Data are presented as boxplot diagrams; the whiskers represent the minimum and maximum values, the dots show single points. Data were analyzed using repeated measures 2-way ANOVA followed by Sidak´s multiple; SAH: n = 5, sham: n = 5; * p < 0.05; dotted line: minimum detectable dose 6.1ng/ml.

Fig 5

Western blot analysis of TNF-α (A) and IL-6 (B) in 3 different brain regions Upper part: representative protein bands detecting TNF-α or IL-6. Lower part: box plots of quantitative analysis from SAH (black) and sham (grey); data were analysed using 2 way ANOVA followed by Tukey´s (within groups) or Sidak´s (between groups) multiple comparisons tests. brain tissue was harvested after euthanasia at the end of the recording phase at 6h. * p < 0.05; ** p < 0.01.