Notwithstanding employment of some genetically based models of PD in rodents and invertebrates, two main experimental animal models of PD have been widely used: (1) the selective degeneration of DA neurons with the neurotoxins, such as N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, [37, 38]) or 6-hydroxydopamine (6-OHDA, ) and (2) the induction of degenerative changes of DA neurons through precise transection of the nigrostriatal afferent fibers . The toxicity of MPTP and 6-OHDA is believed to result from inhibition of complex I of the mitochondrial electron transport chain  and could lead to acute and almost complete DA neuronal cell death within a short time (1-3 days). However, the transection of the adult rat MFB leads to different degrees and rates of DA neuronal death. Our previous studies showed that a time-dependent, progressive loss of DA neurons is the main characteristic of the MFB transection model and this surgical approach permitted axotomized neurons to live for a significant length of time in situ; the statistically significant decrease of DA neurons was observed from 7 days post-transection and about 40% of axotomized DA neurons survived till 70 days after MFB transection [26, 42]. Therefore, MFB transection model is more suitable to study the relationship between DA neuronal degeneration and the surrounding environmental effects, such as neuroglial function.
At 7 days after MFB transection, the protein level of TH in the ipsilateral midbrain tended to decrease compared to the contralateral side, but this decrease was not significant by western blot assay (Figure 1). This result was not consistent with our previous reports: at this time point, when measured by unbiased stereological method, the number of TH-ir neurons in the ipsilateral SN decreased significantly about 10% [26, 42]. This discrepancy could be explained by the difference in methods used. In contrast to the stereological method which confined the region of interest to the SN, in western blot assay, the used tissue block included the ventral tegmental area (VTA) as well as SN. Therefore, the TH protein in the VTA might be summed up in western blot assay and could mask the decrease of TH protein level in the SN. The possibility also cannot be ruled out that the remaining DA neurons in the SN produce more TH protein to compensate the loss of neighboring DA neurons. By and large, the total amount of TH protein was decreased as time elapsed, which means that progressive DA neuronal death occurred following MFB transection.
The precise mechanism of axotomized DA neuronal death is poorly understood, and it might be different from other parkinsonian animal models, because MPTP associated death is TUNEL-positive [43, 44], while MFB transection-induced cell death is TUNEL-negative [45, 46]. Therefore, some authors argued that the cell death mechanism of axotomized DA neurons is entirely necrotic [45, 46]. However, we have presented some different opinions that the mechanism of nigral cell death after axotomy is another type of apoptosis, such as apoptosis-like caspase-independent apoptosis [26, 47]. The latter possibility is suggested by the findings that DAPI staining showed apoptocic figures (i.e. chromatin clumps) in axotomized DA neurons . And many TH-ir neurons, undergoing various steps of consecutive neurodegenerative changes in the ipsilateral SN, retained phosphorylated c-Jun and activating transcription factor 3 in the condensed or fragmented nuclei . It is widely accepted idea that c-Jun phosphorylation triggers the cascades of programed cell death in the injured dopaminergic neurons .
As the gradual DA neuronal death went on, to clear the resultant cell debris and dead or dying neurons from the CNS parenchyma, microglial activation was noted in the ipsilateral SN. Morphologically activated microglia (with large soma as well as shorten and thicken processes) were crowded and they actively expressed MHC class II (OX6-ir) and showed phagocytic characteristics (ED1-ir) (Figure 1). Moreover, these activated microglia produced ROS (O2-and O2
-derived oxidant) and showed a close spatial relationship with collateral TH-ir DA neurons (Figure 2). In this study, hydroethidine (also known as dihydroethidium) was adopted to detect ROS production in situ. Hydroethidine is easily taken up by living cells and shows blue fluorescence. Within the living cell, hydroethidine can be directly oxidized in the presence of ROS, such as O2-, and then, it is converted to red fluorescent ethidium, which in turn is trapped in the cells by intercalation into DNA [32, 49].
Microglial activation involves characteristic morphological transformation from resting to the activated state, and activated microglia release ROS as well as various proinflammatory and neurotoxic factors such as tumor necrosis factor-alpha, interleukin-1beta and so on. [2–5, 50]. The ROS can cross cell membranes easily and induce neuronal death by causing oxidative damage to cellular components . Actually, activated microglia in culture secrete large amounts of H2O2 and NO in a process known as 'respiratory burst’, and can directly damage cells and lead to neuronal cell death . The potential clinical significance of this process has also been highlighted by a postmortem study of PD patients, which showed evidence of accumulation of activated microglia and oxidative modifications of proteins in the SN . It is also well known that DA neurons in the SN possess reduced antioxidant capacity, such as accumulation of total iron and low intracellular glutathione level [54, 55], which render DA neurons more vulnerable to oxidative stress, relative to other cell types. Moreover, the number of microglia is higher within the mesencephalon compared with other brain areas . Taken together, these findings strongly suggest that the release of ROS from activated microglia could increase neurotoxicity to adjacent DA neurons and might contribute to neurodegeneration.
To confirm the role of activated microglia in DA neuronal degeneration, we implanted activated microglia into the SN treated with MFB transection and elucidate whether DA neuronal loss is accelerated. The most popularly used microglial cell line is BV-2 but its application is limited. For instance, BV-2 cells do not give reliable results in chemokine expression and release following LPS treatment compared with primary microglial cells. Moreover, it is natural that microglial cell line cannot help having different biological characteristics since its genetic immortalization might significantly affect its biology [57, 58]. Therefore, in this study, we isolated adult primary microglia from rat brain 7 days after MFB transection.
Previous microglia isolation methodologies generally utilize whole brain regions. However, to utilize whole brain seems to be inadequate. The brain microenvironment is extremely dynamic depending on its distinct anatomical regions and the local neurochemical milieu . Accordingly, the microglial phenotype and activation state, especially under pathological conditions, also have no choice but to show heterogeneity and brain region specificity . The phenotypic diversity displayed by CNS microglia also reflects a functional diversity . Thus, to isolate microglia within interesting brain anatomical loci is thought to be important to elucidate the exact role of microglia in vivo. Therefore, in this study, we isolated activated microglia from the adult rat midbrain of which MFB had been transected 7 days before, and implanted the activated microglia into the ipsilateral SN of the other rat directly of which MFB also had been transected 7 days ago. To isolate microglia from the SN region, we adapted Brewer and Torricelli’s protocols which had been developed to isolate and culture adult rodent neurons . We could isolate highly enriched, quiescent microglia from adult rat SN. Other CNS cell types such as astrocytes and neurons were almost completely excluded (Figure 3). However, the exact identity of Hoechst-positive spots which were not stained with markers used in this study (OX42, NeuN, and GFAP) was obscure. It may be that some nuclei of microglia were not properly stained with OX42. Alternatively, it is possible that the nuclear debris was merely detected with Hoechst stain. In contrast to quiescent microglia isolated from normal rat SN, microglia isolated from the ipsilateral SN showed functionally activated phenotype possessing strong MHC class II and iNOS immunoreactivity (Figure 3). These results mean that the isolation procedure may not alter the microglial immunophenotypes.
We directly transplanted the activated microglia into the ipsilateral SN. So, the mechanical damage caused by the inserted syringe may induce the additional innate microglial activation. As a result, the ipsilateral SN, where the activated microglia were transplanted and treated with MFB transection 7 days ago, has three types of activated microglial cells. The first is innate activated microglia induced by MFB transection. The second is exogenous activated microglia transplanted from other brains. And the third is innate activated microglia induced by syringe insertion. The effect of the first and the third can be exclusory, because the sham controls had been also received the same surgical procedures. Therefore, we could assess the effect of exogenous activated microglia only, and concluded that the transplantation of exogenous activated microglia accelerates the axotomized DA neuronal death significantly (Figure 4).
Tuftsin fragment 1-3, a human IgG derived tripeptide, Thr-Lys-Pro, was first identified by Auriault and colleagues . Since its potential role as a macrophage inhibitory factor was demonstrated, tuftsin fragment 1-3 has also been used as a microglia inhibitory factor in other studies, although its precise mechanism is unknown [28, 63, 64]. For example, Thanos and colleagues demonstrated that a single intravitreal injection of tuftsin fragment 1-3 inhibited phagocytic activity of microglia and increased the number of regenerating axons of retinal ganglionic cells by two to threefold . Tuftsin fragment 1-3 also decreased ROS production from activated microglia, resulting in a promising reduction of brain edema and tissue damage and attenuation of functional deficits in ischemic animal brains .
In this study, we used tuftsin fragment 1-3 as a microglial inhibitory factor to investigate the role of activated microglia in the pathogenesis of axotomized DA neuronal degeneration. In tuftsin fragment 1-3 treated animal, the number and intensity of OX6-ir and ED1-ir activated microglial cells were dramatically decreased and the survival rate of ipsilateral DA neurons was improved about 20% compared with vehicle treated ones (Figure 5). As a consequence, tuftsin fragment 1-3 was proved to be an effective inhibitor of microglial activation and this inhibitory effect was thought to contribute to the increased survival rate of axotomized DA neurons.