Original article
Quantitative analysis of activated microglia, ramified and damage of processes in the frontal and temporal lobes of chronic schizophrenics

Communicating author:
doc. dr hab. Teresa Wierzba-Bobrowicz, Department of Neuropathology, Institute of Psychiatry and Neurology, Sobieskiego 9, 02-957 Warsaw, Poland, e-mail: bobrow@ipin.edu.pl





Introduction
In the central nervous system (CNS), microglial cells make 15-20% of the total number of cells [7,36]. Although microglial cells appear prior to the brain vascularisation and monocytes, it is generally thought that they originate from the hematopoidal line [7,23]. From the eighth week of intrauterine life, they emerge in all CNS structures evenly distributed in the grey and white matter.
Microglia exhibit an extensive morphological plasticity. Activated microglia may assume different morphological forms, depending on morphoarchitecture of their structure, in which they occur, on the function performed in a given moment as well as on pathological agent exerting its effect [23,24]. Activated microglial cells most frequently assume the form of buscy microglia and/or ramified microglia (RM). However, spindle, rod and ameboid cells of different shapes, cytoplasm abundance, thickness, and the number of processes are also visible [19,26].
Microglia are also multifunctional cells. During the CNS development, they play a crucial role in eliminating necrotic neurons in the mechanism of apoptosis. They are also involved in vasculogenesis, migration and functional differentiation of neurons [7,23]. In the mature CNS, they primarily play the role of immunocompetent cells equipped with the fagocyting ability. The recent reports suggest their new role of multipotential stem cells [16,36]. Under pathological conditions, microglial cells are rapidly activated, which is mostly manifested by showing on their surface the expression of the major histocompatibility complex class II (MHC II), and also by their proliferation and appearance in new/different morphological forms [3,4,26].
Proliferation of microglia has also been reported in schizophrenia although its etiopathogenesis has not yet been conclusively elucidated [21,18,35]. Their proliferation is particularly observed in structures whose abnormal functioning suggests clinical and neurochemical symptoms [1,2,28,30]. A diminished volume of individual structures of the brain, most frequently linked functionally, as well as the reduced number of neuronal processes, axons, dendrites, and synapses, and the decreased level of neurotransmitters have already been described [6,28]. Our previous study showed the presence of activated microglial cells, ramified and damage of processes, which exhibited some degenerative traits in both frontal and temporal lobes. These structures are linked with each other by inhibitory and excitatory projections via mammilary bodies and anterior thalamus [8,10,27]. These changes suggested a secondary damage of the former normal, activated microglial cells in the course of chronic schizophrenia [31,35], which indicates their involvement in exacerbation of structural changes in the long-term morbid process. The aim of the present study was to perform a quantitative analysis of activated microglia, normally ramified and exhibiting degenerative traits, in both frontal and temporal lobes in chronic schizophrenics. The quantitative assessment of both morphological forms of microglia may contribute to better understanding of agents participating in pathogenesis of schizophrenia.

Material and methods
The quantitative analysis was performed on serially cut slices, derived from the temporal lobe (gyrus temporal inferior, Brodmann’s area 20) and the frontal lobe (gyrus cinguli, Brodmann’s area 24). The study material was obtained from 9 brains of female chronic schizophrenics (Table I). Control brains were obtained from 6 patients of the same age group and free from neurological illnesses with extracerebral causes of their deaths (Table II).
The brains were fixed in 4% paraformaldehyde in 0.1 M phosphorane- buffer saline, pH 7.4. Frontal and temporal lobe slices were cut serially at 8 µm. Expression of MHC II was visualised immunohistochemically, using anti-human HLA-DP, DQ, DR (DAKO 1:50). The quantitative analysis was performed on three consecutive preparations of each series. Microglial cells were counted in 10 not overlapping regions of 0.076 mm2 (Neofluar 40x, Axiophot Zeiss), localised axially along five segments of 3.5 mm, running from the surface deep into the cortex at intervals of 0.5 mm (Fig. 1). The values of arithmetic mean (±SE) of the identified numbers are given in summary tables. The nonparametric Mann-Whitney U test was used to assess quantitative differences in the distribution of microglia in the studied regions of the brain. Statistical analyses were performed with STATISTICA 6.5 Programme (Stat Soft USA).

Results
The quantitative analysis of microglial cells was performed (neocortex) on the frontal lobe (gyrus cinguli, Brodmann’s area 24) and the temporal lobe (gyrus temporal inferior, Brodmann’s area 20). The increase in the number of activated microglial cells, ramified and with damaged processes (from their shortening to their complete lack (DM)), was observed in both structures (Table III, Fig. 1).
In gyrus cinguli, the majority of microglia, both RM and DM, was localised in the submeningeal region (0.35), where the numerical values of both morphological forms were comparable (Fig. 2). In the layers localised deep into the cortex (0.7-3.5 mm), the number of RM cells was 2-3 times higher than that observed in the control material, whereas the amount of microglia damage of processes was 3-7 times higher than that in the cortex of control brains. In the regions between 0.7 and 3.5 mm, microglial cells with damaged processes largely outnumbered RM cells.
In gyrus temporal inferior in the subcortical layer (0.35), the numbers of RM and DM were similar and only slightly exceeded the control values (Fig. 3). The number of activated microglial cells increased with calculations descending deeper into the cortex, from 0.7 to 3.5 mm (Table IV). In those regions of the cortex, DM cells were definitely superior in numbers to RM cells. Only in the deepest regions (3.15-3.5 mm) the numbers of RM and DM cells were similar, however, they substantially exceeded the control values. The control results showed that the number of both RM and DM cells was over twice as high in the temporal lobe as that in the frontal lobe (Tables V and VI).
Discussion
The quantitative analysis of ramified microglia and microglia with diversified damage to processes, which exhibited the expression of histocompatibility locus antigens class II (HLA II) for a specific immunohistochemical marker of activated microglia, showed a significant increase in the pool of microglial cells in the brain of chronic schizophrenics. This form of activity was observed in both morphological forms of microglia (RM and LM) in the frontal lobe as well as in the temporal lobe. It seems that the possibility of producing HLA II at the genic and molecular levels is not impaired by agents responsible for schizophrenia generation, duration of the disease, or long-term use of psychotropic drugs [11,21]. There is a number of agents that can influence proliferation and activation of microglial cells in the frontal and temporal lobes of patients with chronic schizophrenia [24,25,29]. They may include unknown causes possibly fundamental for etiopathogenesis of schizophrenia (e.g., neurodevelopmental disorders, infections), structural changes (e.g., defects of neuronal and glial cells, loss of axons and dendrites), and the decline in highly energetic compounds (ATP below 30-50 µM), or disturbances at the level of neurotransmitters [1,5,6,13,20,21]. On the other hand, activated microglia show the ability to generate bioactive, protective (e.g., plasminogen, IL-6) and structural agents in the CNS. The majority of microglia devoid of processes were characterised by more or less advanced degenerative traits. Abnormalities in toxic molecules such as IL-1βb or TNF-α, which may be fundamental to structural and functional damages to neurones and oligodendroglia in chronic schozophrenia, may be regarded as agents able to trigger a cascade transforming RM into DM [12,17].
In all studied regions of both temporal and frontal lobes, activated DM cells outnumbered RM cells. In the brain of schizophrenics, the DM frequently multiplied RM values, especially in the frontal lobe. Only in the submeningeal region (0.35 mm) (first cortical layer), the numbers of both morphological forms of microglia were balanced, which may suggest that this region is structurally privileged in terms of the ”inflow” and/or the absence of effects exerted by agents involved in the transformation of RM into DM. Cerebral meninges like periventricular region and vessels are described as places where microglia penetrate the CNS [7,23,33,34]. Moreover, in the first layer of neocortex, intra-layer links are not expanded as this layer is mostly composed of Calaj cells with processes running in parallel to meninges and astroglial cells [32].
Morphological forms of cerebral microglia/ /macrophages with a limited number of processes or completely devoid of them, frequently exhibited the ability to phagocytose damaged CNS structural fragments. The majority of forms of microglia devoid of processes were characterised by more or less advanced degenerative traits. The disturbed calcium metabolism (Ca2+) and energetic balance as well as generation of antibodies described in schizophrenia (e.g, antibrain antibodies, antibodies to basic myelin-protein, antibodies to a brain glycoprotein fraction) may be regarded as agents able to trigger a cascade transforming RM into DM [9,11,15,20]. There were quantitative differences in morphological forms of microglia (RM and DM) between the frontal and temporal lobes as well as between the studied individual regions localised in different layers of neocortex. The observed differences could be not only due to different advancement of cellular and molecular changes, and the effect of pathological agents, but also due to altered functioning of inter-neuronal and inter-structural links [14,22,32].
The present study may indicate a pivotal role of microglial cells in repair processes and/or generation and development of structural, molecular and functional changes in chronic schizophrenia. It also suggests that a substantial amount of microglia may be subjected to considerable damage in the course of chronic schizophrenia.

References
1. Andreasen NC. Schizophrenia: the fundnamental questions. Brain Res Brain Res Rev 2000; 31: 106-112.
2. Antonova E, Sharma T, Morris R, Kumari V. The relationship between brain structure and neurocognition in schizophrenia: a selective review. Schizophr Res 2004; 70: 117-145.
3. Bertrand E, Lechowicz W, Szpak GM, Lewandowska E, Dymecki J, Wierzba-Bobrowicz T. Limbic neuropathology in idiopathic Parkinson’s disease with concomitant dementia. Folia Neuropathol 2004; 42, 3: 141-150.
4. Bertrand E, Lechowicz W, Szpak G, Lewandowska E, Dymecki J, Kosno-Kruszewska E, Wierzba-Bobrowicz T. Degenerative axonal changes in the hippocampus and amygdala in Parkinson’s disease. Folia Neuropathol 2003; 41, 4: 197-207.
5. Corfas G, Roy K, Buxbaum JD. Neuregulin 1-erbB signaling and the molecular/cellular basis of schizophrenia. Nat Neurosci 2004; 7, 6: 575-580.
6. Cotter D, Mackay D, Beasley C, Kervin R, Everall I. Reduced glial density and neuronal volume in major depressive disorder and schizophrenia in the anterior cingulate cortex (abstract). Schizophr Res 2000; 41: 106.
7. Cuadros MA, Navascues J. The origin and differentiation of microglial cells during development. Prog Neurobiol 1998; 56: 173-189.
8. Dehaene S, Artiges E, Naccache L, Martelli C, Viard A, Schurhoff F, Recasens Ch, Martinot ML, Leboyer M., Martinot J-L. Conscious and subliminal conflicts in normal subjects and patients with schizophrenia: The role of the anterior cingulate. Proc Natl Acad Sci USA 2003; 100 (23): 13722-13727.
9. DeLisi LE, Weber R, Pert C. Anti-brain antibodies in psychotic patients: Review and perspectives. Biol Psychiatry 1985; 20: 110-119.
10. Galliant J, Mulert Ch, Bajbouj M, Herrmann WM, Schunter J, Senkowska D, Moukhtieva R, Kronfeldt D, Winterer G. Frontal and Temporal Dysfunction of Auditory Stimulus Processing in Schizophrenia. Neuroimage 2002; 17: 110-127.
11. Ganguli R, Brar JS, Chengappa KNR, Yang ZW, Nimgaonkar VL, Rabin BH. Autoimmunity in schizophrenia: A review of recent findings. Ann Med 1993; 25: 489-496.
12. Imai Y, Kohsaka S. Intracellular Signaling in M-CSF-Induced Microglia Activation: Role of Iba1. Glia 2002; 40: 164-174.
13. Inoue K. Microglial Activation by Purines and Pyrimidines. Glia 2002; 40: 156-163.
14. Kalla R, Bohatschek M, Kloss CU, Krol J, Von Maltzan X, Raivich G. Loss of microglial ramification in microglia-astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems. Glia 2003; 41: 50-63.
15. Lidow MS. Calcium signaling dysfunction in schizophrenia: a unifying approach. Brain Res Brain Res Rev 2003; 43: 70-84.
16. Liva SM, De Vellis J. IL-5 induces Proliferation and Activation of Microglia via an Unknown Receptor. Neurochem Res 2001; 26 (6): 629-637.
17. Minghetti L, Levi G. Microglia as effectors cells in brain damage and repair: focus on prostanoids and nitric oxide. Prog Neurobiol 1998; 54: 99-125.
18. Munn NA. Microglia dysfunction in schizophrenia: an integrative theory. Med Hypotheses 2000; 54 (2): 198-202.
19. Nelson PT, Soma LA, Lavi E. Microglia in disease of the central nervous system. Ann Med 2002; 34: 491-500.
20. Petersen MA, Dailey ME. Diverse Microglial Motility Behaviors During Clearance of Dead Cells in Hippocampal Slices. Glia 2004; 46: 195-206.
21. Radewicz K, Garey LJ, Gentleman SM, Reynolds R. Increase in HLA-DR Immunoreactive Microglia in Frontal and Temporal Cortex of Chronic Schizophrenics. J Neuropathol Exp Neurol 2000; 59 (2): 137-150.
22. Ranasinghe S, Bolsover S. Microglial Calcium Responses to Platelet-Activating Factor are Inhibited by Analogue CAS 99103-16-9 and Dihydropyridine PCA 4248 but Not by Ginkgolide A. Basic Clin Pharmacol Toxicol 2004; 95: 87-91.
23. Streit WJ. Microglia as Neuroprotective, Immunocompetent Cells of the CNS. Glia 2002; 40: 133-139.
24. Sugama S, Wirz SA, Barr AM, Conti B, Bartfai T, Shibasaki T. Interlukin-18 null mice show diminished microglial activation and reduced dopaminergic neuron loss following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. Neuroscience 2004; 451-458.
25. Sweet RA, Bergen SE, Sun Z, Sampson AR, Pierri JN, Lewis DA. Pyramidal Cell Size Reduction in Schizophrenia: Evidence for Involvement of Auditory Feedforward Circuits. Biol Psychiatry 2004; 55: 1128-1137.
26. Szpak GM, Lechowicz W, Lewandowska E, Bertrand E, Wierzba-Bobrowicz T, Gwiazda E, Schmidt-Sidor B, Dymecki J. Neurones and microglia in central nervous system immune response to degenerative processes. Part I: Alzheimer’s disease and Lewy body variant of Alzheimer’s disease. Quantitative study. Folia Neuropathol 2001; 39, 3: 181-192.
27. Takahashi T, Suzuki M, Zhou S-Yu, Hagino H, Kawasaki Y, Yamashita I, Nohara S, Nakamura K, Seto H, Kurachi M. Lack of normal gender differences of the perigenual cingulate gyrus in schizophrenia spectrum disorders. A magnetic resonance imaging study. Eur Arch Psychiatry Clin Neurosci 2004; 254: 273-280.
28. Tamminga CA, Vogel M, Gao XM, Lahti AC, Holcomb HH. The limbic cortex in schizophrenia: focus on the anterior cingulate. Brain Res Brain Res Rev 2000; 31: 364-370.
29. Taylor DL, Diemel LT, Pocock JM. Activation of microglial group III metabotropic glutamate receptors protects neurons against microglial neurotoxicity. J Neurosci 2003; 23 (6): 2150-2160.
30. Thopmson PM, Egbufoama S, Vawter MP. SNAP-25 reduction in the hippocampus of patients with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2003; 27: 411-417.
31. Wank R. Schizophrenia and other mental disorders require long-term adoptive immunotherapy. Elsevier Science 2002; 59 (2): 154-158.
32. Watts J, Thomson AM. Excitatory and Inhibitory Connections show Selectivity in the Neocortex. J Physiol, in press 2004.
33. Wierzba-Bobrowicz T, Schmidt-Sidor B, Gwiazda E, Lechowicz W, Kosno-Kruszewska E. Major histocompatibility complex class II expression in the frontal and temporal lobes in the human fetus during development. Folia Neuropathol 2000; 38 (2): 73-77.
34. Wierzba-Bobrowicz T, Kosno-Kruszewska E, Gwiazda E, Lechowicz W. Major histocompatibility complex class II (MHC II) expression during the development of human fetal cerebral occipital lobe, cerebellum, and hematopoietic organs. Folia Neuropathol 2000; 38, 3: 111-118.
35. Wierzba-Bobrowicz T, Lewandowska E, Kosno-Kruszewska E, Lechowicz W, Pasennik E, Schmidt-Sidor B. Degeneration of microglial cells in frontal and temporal lobes of chronic schizophrenics. Folia Neuropathol 2004; 42, 3: 157-165.
36. Yokoyama A, Yang L, Itoh S, Mori K, Tanaka J. Microglia, a Potential Source of Neurons, Astrocytes, and Oligodendrocytes. Glia 2004; 45: 96-104.