Krzysztof Janeczko

Morphology of reactive microglia in the injured cerebral cortex. Fractal analysis and complementary quantitative methods.

The present study focuses on application of quantitative methods measuring differences between particular morphological types of microglial cells as well as between their proliferating and non‐proliferating examples. On the basis of subjective classification, microglial cells of three morphological types (ramified, hypertrophied and bushy) were selected from the neocortex of injured rat brain. Thereafter, the morphological complexity of each cell was assessed by calculation its fractal dimension as well as its form factor, convexity, ramification factor and solidity. The fractal dimension seemed a good parameter for detecting small changes in the space‐filing capacity of cells, for example, it shows differences between ramified cells from control and injured brains. This measure seemed insensitive to some aspects of cell morphology. To obtain precise quantification of observed changes other morphological parameters had to be applied. Proliferating and non‐proliferating microglial cells displayed significant differences in their solidity and ramification factors, but not in fractal dimension and convexity. The results indicated that proliferating microglia were more massive and less‐ramified but they did not reduce their spatial complexity. J. Neurosci. Res. 63:90–97, 2001.

The proliferative response of astrocytes to injury in neonatal rat brain. A combined immunocytochemical and autoradiographic study

Astrocyte response to unilateral injury of the cerebral hemisphere in newborn rats was studied both autoradiographically and immunocytochemically. Within the lesion area zones of increased number of labelled astrocytes were delimited. They were considered as evidence of reactive astrocyte proliferation in the rat brain injured at birth.

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: (1) astrocytes co‐expressing glial fibrillary acidic protein (GFAP) and vimentin (2) astrocytes expressing only GFAP (3) astrocyte‐like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP‐positive astrocytes and of those co‐expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP‐positive astrocytes, 67.2% were also vimentin‐positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte‐like vimentin‐positive cells were located closest to the lesion margins. In comparison, the astrocytes co‐expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP. This was considered as an argument against a hypothesis that reactive astrocyte division induces a two‐stage increase in the cytoskeletal protein level in which synthesis of vimentin precedes that of GFAP.

Spatiotemporal patterns of the astroglial proliferation in rat brain injured at the postmitotic stage of postnatal development: a combined immunocytochemical and autoradiographic study

Proliferative response of astrocytes to unilateral injury of the cerebral hemisphere was investigated in 30-day-old rats using a combination of [3H]thymidine autoradiography and immunocytochemical staining for glial fibrillary acidic protein. At different intervals following injury the animals were injected with [3H]thymidine and the distribution of double-labeled cells was recorded 4 h (short-term experiment) or 40-days (long-term experiment) after each injection. Within the region of the lesion a strong reactive proliferation of astrocytes began as early as 2 h after injury, although, at that time all dividing neuroglial cells were located at a relatively long distance from the lesion margin. Forty days later, however, autoradiographically labeled astrocytes were observed in the center of the lesion. This was regarded as evidence for the ability of astrocytes to migrate towards the site of injury. Maximal reactive proliferation of astroglial cells was observed at day 2 after injury and then reached the postmitotic plateau at about day 8. The cells, while dividing, were mostly GFAP-negative. Therefore, the number of astrocytes produced at particular days after injury, and their contribution to post-traumatic scar formation could be assessed only in the long-term experiment, after the newly formed cells became GFAP-positive.

Quantitative morphological study of microglial cells in the ischemic rat brain using principal component analysis.

Pathogenic stimuli induce alterations in the morphology of microglial cells. We analysed changes in lectin-stained cells on the 1st, 3rd, 7th or 14th day after transient global ischemia. Three areas differing in the degree of microglial reaction were selected for analysis: the upper cerebral cortex, the hippocampal CA1 area, and the hilus of the dentate gyrus. Nine morphological parameters, including fractal dimension, lacunarity, self-similarity range, solidity, convexity and form factor were determined. Then the resultant data were processed using principal component analysis (PCA). We found that the two first principal components together explained more than 73% of the observed variability, and may be sufficient both to describe the morphological diversity of the cells, and to determine the dynamics and direction of the changes. In both hippocampal areas, the transformation to hypertrophied and phagocytic cells was observed, but changes in the hilus were faster than in the CA1. In contrast, in the cortex, a microglial reaction was characterised by an increase in the complexity of processes. The results presented show that the quantitative morphological analysis can be an effective tool in research on the reactive behaviour of microglia and, particularly, in the detection of small and early changes in the cells.

Concomitant up-regulation of astroglial high and low affinity nerve growth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat.

We have examined the effect of global transient cerebral ischemia, evoked in rat by 10 min of cardiac arrest, upon the changes in the cellular expression of two nerve growth factor (NGF) receptors (TrkA and p75) in the hippocampus. We have used immunocytochemical procedures, including a quantitative analysis of staining, along with some quantitative morphological analyses. We have found, under ischemic conditions, a decrease of TrkA immunoreactivity in degenerating CA1 pyramidal neurons and in neuropil. On the other hand, a strong, ischemia-induced up-regulation of TrkA and p75 immunoreactivity was observed in the majority of reactive astroglia population in the adjacent CA1 hippocampal region. The colocalization of the two receptors in the same reactive astroglial cells was evidenced by double immunostaining and further supported by quantitative morphological analysis of TrkA and p75 immunoreactive glial cells. Our data implicate the involvement of NGF receptors in the postischemic regulation of astrocytic function; however, the lack of NGF receptor expression on some astrocytes suggests heterogeneity of astroglia population. Our results also indicate that the lack of neuroprotective action of astroglial NGF induced in the ischemic hippocampus [J Neurosci Res 41 (1995) 684; Acta Neurobiol Exp 57 (1997) 31; Neuroscience 91 (1999) 1027] is not caused by a paucity of NGF receptors but may rather be due to the counteraction of some proinflammatory substances, released simultaneously by glia cells. On the other hand, the up-regulated astroglial TrkA receptor may be an important target for exogenous NGF, which, as previously described [J Neurosci 11 (1991) 2914; Neurosci Lett 141 (1992) 161], exerts a neuroprotective effect in ischemia.

Morphological development of microglia in the postnatal rat brain. A quantitative study.

Morphological transformation of lectin‐positive microglia/macrophages in the developing rat cerebral hemisphere was analysed using quantitative methods. During the first postnatal month, the cells showed increases in their size and fractal dimension accompanied by a simultaneous decrease in their solidity. Regional variations in dynamics of the process indicated the existence of spatio‐temporal developmental gradients within the cerebral hemisphere wall which might correspond with regional patterns of neuronal differentiation. Results of the present study prove that the quantitative methods can be the source of reliable data replacing subjective cell typologies.

Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures

In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10 microm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammons horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958 cm(-1)) as well as conformational changes of proteins (beta-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657 cm(-1) as well as the ratio of the absorbance at 1548 and 1657 cm(-1)) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures.

Long-term changes in postnatal susceptibility to pilocarpine-induced seizures in rats exposed to gamma radiation at different stages of prenatal development.

Summary:  Purpose: To determine whether brains irradiated at different stages of prenatal development also have different postnatal susceptibility to seizures evoked by pilocarpine.

SPATIOTEMPORAL PATTERNS OF MICROGLIAL PROLIFERATION IN RAT BRAIN INJURED AT THE POSTMITOTIC STAGE OF POSTNATAL DEVELOPMENT

Changes in the number and distribution of microglial cells proliferating in response to unilateral injury of the cerebral hemisphere were investigated in 30‐day‐old rats. Twelve hours or 1, 2, 4, or 8 days following the injury the rats were injected with 3H‐thymidine and killed 4 hr later. Brain sections were processed for BSI‐B4 isolectin histochemistry followed by autoradiography. During microscopic observations, four morphological types of lectin‐positive and autoradiographically labeled cells were distinguished: 1) ramified, 2) hypertrophic, 3) bushy, and 4) macrophages. Subsequently, numbers and locations of the cell types within the injury area were recorded at different stages of the experiment. The earliest signs of the proliferative response were displayed 12 hr after injury by ramified microglia. On the first day after injury, those cells represented about 50% of the whole cell population and were spread at relatively the longest distances from the lesion site. During subsequent stages of the response, a considerable reduction of the area occupied by proliferating microglia corresponded with a dramatic quantitative decrease of their ramified fraction and a simultaneous increase of their more advanced reactive forms and macrophages. J. Neurosci. Res. 58:379–386, 1999.