The major experimental aim of this chapter was to develop a method to deliver an aseptic closed-head injury to the CNS. The adoption of a liquid nitrogen-cooled steel rod to administer cortical cryolesion to MT-I/II−/− and wild type mice resulted in a reproducible and well-defined injury site. Using this method, no differences in lesion size were observed between wild type and MT-I/II−/− mice. Significant increases in neuron death and T cell infiltration were apparent in MT-I/II−/− mice, but only at 7 DPI. The fluoro-jade C data show that for wild type mice, the injury has transitioned from a degenerative phase observed at 1-3 DPI, to a regenerative phase by 7 DPI. The macrophage and T cell density in the injury site was highest at 7 DPI suggesting that these cells may influence the recovery from brain injury after the degenerative period has ceased in wild type mice. It would also appear that the injured brain of MT-I/II−/− mice is not transitioning to a regenerative state in the same manner as wild type mice based on the fluoro-jade C and immune cell temporal distribution.
2.4.1 Neuron death and T cell infiltration is altered in MT-I/II−/− mouse brain injury Prolonged neuron death and increased T cell infiltration in the injury site were major differences observed between MT-I/II−/− mice and wild type mice. It is possible that the two phenomena are related because neurons can directly trigger microglial activation in vitro (Sudo et al. 1998) which can lead to the inflammatory processes that promote the infiltration of T cells. Additionally, T cells have been shown to directly cause neuron death in vitro (Giuliani et al. 2003). From the present data it is not possible to determine whether neuron death and T cell infiltration are related in MT-I/II−/− mice but the fact that they both differed from wild type mouse levels at 7 DPI, but no other time points, presents the possibility that the two events are linked. A latent period before increased neuron death in MT-I/II−/− mice compared to wild type mice has been reported previously (Natale et al. 2004). However, increased neuron death in MT-I/II−/− mice has also been observed within 24 hours of brain injury (Penkowa et al. 1999a). One of the criticisms of the study by Penkowa et al. (1999a) is that neuron death was assessed by counting neuron-specific enolase labelled neurons immediately adjacent to the injury border. This approach assumes that neuron numbers in the region of interest were identical in wild type mice and MT-I/II−/− mice before the injury occurred. Because, in their study, the injury site was larger in MT-I/II−/− mice, the border of the injury site would be at different depths in wild type mice and MT-I/II−/− mice and
of the cortex. It is well known that there are large differences in the density and distribution of neurons in different layers of the cortex (DeFelipe et al. 2002). The present study avoids these problems because the injury size did not differ between the strains at any time point and neuron death was measured directly.
In light of this, the important question becomes: why do MT-I/II−/− mice have an increased injury size in dry ice induced cortical lesions compared to the liquid nitrogen induced lesion used in the present study? The cryolesion model presented here consists of a 6 second freeze with a steel rod cooled to approximately −196°C which is sufficient to cause damage to neural tissue but the temperature of the cortex is only lowered momentarily, whereas cryolesion by dry ice (−78°C) requires application to the skull for 90 seconds (Penkowa and Moos 1995). The dry ice cryolesion induced a high degree of haemorrhage which was determined by both a visible accumulation of haemoglobin in the injury site and immunohistochemical staining for extravasation of albumin within 30 minutes of injury (Penkowa and Moos 1995). Albumin extravasation was not investigated in the present study, but substantial numbers of red blood cells in the injury site were not found until 3 DPI (most evident in figure 2.4). The dry ice cryolesion also produced a lesion with an irregular shape (Penkowa and Moos 1995) which may affect the ability to compare injury size in histological sections. In the present study, the affected surface of the cryolesioned brain was consistently circular in shape (personal observation) and had some bruising but the haemorrhaging was not to the extent observed by Penkowa and Moos (1995). Therefore the two methods of administering cryolesion injury differ considerably and therefore the sequelae of the injury is also likely to differ which may explain differences in the activation of the macrophage response to the injury.
The level of T cell infiltration into MT-I/II−/− mouse cryolesions has not been compared to wild type mice before. However it has been shown that transgenic over-expression of MT-I in mice leads to decreased T cell infiltration into the CNS after cryolesion or kainic acid injection to the hippocampus (Giralt et al. 2002, Penkowa et al. 2005). Systemic injection of MT-II protein after brain injury causes decreases in T cell infiltration in wild type and MT-I/II−/− mice (Giralt et al. 2002, Penkowa et al. 2006b) which might suggest that extracellular MT-I/II can affect the T cell response to brain
required to enter the brain to affect the infiltration of T cells into the injury site. However, it has been shown that injection of MT into mice and rats alters the numbers of macrophages and T cells in the spleen and bone marrow (Giralt et al. 2002, Penkowa and Hidalgo 2000). Increased plasma concentration of MT has been observed in patients with traumatic brain injury (Kukačka et al. 2006), which gives credence to the hypothesis that extracellular MT could influence the progression of brain injury indirectly by modulating the immune system.
2.4.2 Injury-site neutrophil and macrophage numbers are not altered in MT-I/II−/− mice Comparison of neutrophil infiltration into the injured CNS of wild type and MT-I/II−/− mice has not been previously reported. Because neutrophils showed the lowest infiltration cumulatively over the 7 day experimental period and were mostly absent from injury sites at 7 DPI, they probably do not contribute to the altered levels of cell death and T cell infiltration in MT-I/II−/− mice at 7 DPI. The finding that numbers of macrophages in the injury site did not differ between the MT-I/II−/− mice and wild type mice is in contrast to previous studies that show higher numbers in MT-I/II−/− mice after injury (Penkowa et al 1999a, Potter et al. 2007, Potter et al. 2009). However, in experimental brain injury induced by systemic 6-aminonicotinamide injection and hippocampal injection of kainic acid, numbers of cells expressing macrophage markers in the CNS of MT-I/II−/− mice were lower than wild type mice (Penkowa et al. 1999b, Carrasco et al. 2000). Therefore different injury models can vary greatly in the response they elicit from microglia and macrophages.
An important question regarding the technique of cell counting for macrophages using immunohistochemistry is whether the technique adequately identifies all microglia and monocytes present, or labels only a subset of the most activated cells in the injury site. Because microglia express macrophage markers at lower levels when quiescent (Denker et al. 2007, Sedgwick et al. 1991, Stirling and Yong 2008), the observations of differences in macrophages in previous studies may represent different activation states of microglia in MT-I/II−/− mice rather than differences in microglial cell density. In the present study sufficient numbers of cells could not be isolated from the cryolesion injury site to assess microglia/macrophage activation by flow cytometry (data not shown). However all macrophage counts were confined to the injury site in the present study. Given the fact that the injury site is almost devoid of cells at 1 DPI, any cells
migration requires activation, the cells found in the injury site have all undergone activation and are likely to be expressing macrophage markers strongly.
2.4.3 Conclusion
The cryolesion injury model developed for assessing the response to injury induces a reproducible lesion with a substantial infiltration of immune cells. The increase in T cell infiltration into the lesion site at 7 DPI in MT-I/II−/− mice is consistent with several other studies and is of interest due to the potential for MT-I/II to affect brain injury by modulating T cell responses. It warrants mention that in the studies reviewed by Donnelly and Popovich (2008), the function of the immune system may persist for some time after the 7 DPI time point used in these experiments and it remains possible that additional differences in the immune function of MT-I/II -/- mice may become apparent as the brain injury progresses further. Subsequent chapters of this thesis seek to elucidate some of the mechanisms by which MT-I/II−/− mice have an altered immune response to brain injury.