Original article
                     Calbindin positive Purkinje cells in the pathology of human 
                     cerebellum occurring at the time of its development
                                                    1                     1,2
                     Milena Laure-Kamionowska , Danuta Maślińska
                     1Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 
                     2
                     Department of Pathophysiology, Medical University of Warsaw, Warsaw, Poland 
                     Folia Neuropathol 2009; 47 (4): 300-305
                     Abstract
                     Development of cerebellum continues over an extremely long period of time extending from the early embryonic phase 
                     until the first postnatal years. During an extended time of maturation the cerebellum is vulnerable to harmful agents. 
                     A group of cytoplasmic proteins that may protect cells against injury are the calcium binding proteins, among others 
                     calbindin. The distribution of this protein is not well known in cerebellar pathology, thus in the present study the 
                     localisation and appearance of calbindin expressing Purkinje cells in different pathological conditions occurring at the 
                     time of cerebellar development was examined. The investigations were carried out on human maturing cerebellar cortex 
                     (age range 30 gestational weeks – 2 years) of cases with paraneoplastic cerebellar degeneration and cerebellar neuronal 
                     migration disturbances. The Purkinje cells located in cerebellar heterotopias and dysgenesias were morphologically 
                     well developed and strongly immunostained with calbindin antibody. In paraneoplastic cerebellar degeneration the 
                     progressive decrease of calbindin content and disintegration of Purkinje cells were observed. Our results show that 
                     intrauterine harmful agents that disturb migration of the cerebellar neurons do not affect the content of calbindin in 
                     misoriented neurons and that this protein may play a role in development of Purkinje cells located in heterotopias and 
                     cerebellar dysgenesias. The progressive decrease of calbindin content in the Purkinje cells undergoing degeneration and 
                     death during paraneoplastic changes in the cerebellum also supports the hypothesis that this protein is very important 
                     component of intracellular homeostasis in cerebellar neurons.
                     Key words: CalbindinD28k, cerebellum, Purkinje cells, development, human, pathology.
                     Introduction                                                      leads to localised cortical dysplasia and heterotopias 
                                                                                       [7,11].  In  general, cerebellar heterotopias exist in the 
                         Development of cerebellum continues over an extre-            white matter of the hemispheres and consist of nests 
                     mely long period of time extending from the early em-             of large cells resembling Purkinje cells, which may have 
                     bryonic phase until the first postnatal years [1]. Agents         an admixture of granular neurons. When an agent acts 
                     that interrupt normal growth may have different effects           after the Purkinje cells have matured but granular cells 
                     depending on the stage of development. During an ear-             are still dividing and migrating diffuse lesions affecting 
                     ly development focal disruption of neuronal migration             the entire granular layer may appear [8]. 
                     Communicating author:
                     Milena Laure-Kamionowska, Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, Polish 
                     Academy of Sciences, Pawinskiego 5, 02-106 Warszawa, Poland, Phone number: +48 22 608 65 03, Fax nummber: +48 22 608 65 02, Email: 
                     milenak@cmdik.pan.pl.
                     300                                                                                                Folia Neuropathologica 2009; 47/4
                                                                                                             Calbindin neurons in human pathology
                       After  birth  cerebellar  injury  usually  consists  of    30, 32, 34, 36 gestational weeks, term newborns, 2, 
                    diffuse loss of Purkinje cells with less conspicuous          3, 6 months and one year infants. 
                    damage of granular cells. The cerebellar white matter             Fourteen brains of age matched controls were 
                    show evidence of secondary degeneration and the               used in the study. Ten cases aged 30, 32, 34, 36, 
                    deep cerebellar nuclei are only rarely involved. It has       38 gestational weeks and term were found among 
                    been estimated that cerebellar degeneration is most           newborns  delivered  after  normal  pregnancies.  All 
                    often associated with neoplastic diseases [4].                of them died due to acute perinatal pathology and 
                       A group of cytoplasmic proteins that may protect           were not put on controlled respiration. Four infants 
                    cells against injury are the calcium binding proteins.        aged 2 months, 6 months, one year and two years 
                    This group includes such proteins as calretinin, cal-         died after short, severe illnesses or sudden death. 
                    bindin D-28K and parvalbumin [14]. All of them are            Death was caused by accidental intoxication, aspira-
                    characterised by the presence of a variable number            tion, car accidents. These children were not treated 
                    of helix-loop-helix motives, which bind calcium ions          at all or only a few hours by controlled respiration. 
                    with high affinity and are considered to be cyto-             After autopsy all brains were fixed in formalin, em-
                    solic calcium buffers that may modify the spatio-             bedded in paraffin, and stained with haematoxylin 
                    temporal aspects of calcium transients in cells [12].         eosin and cresyl violet to identify morphology. These 
                    These proteins are particularly enriched in specific          brains were diagnosed as nonpathological.
                    cerebellar neurons but distribution each of them in               Neuropathological  examination  was  performed 
                    these neurons is different [3]. The distribution of           on representative slides from the cerebellar hemi-
                    all above mentioned proteins that may protect or              spheres and vermis at several levels.
                    defend human cerebellum against different harmful                 Sections of cerebellum were incubated in prima-
                    agents is not well known in cerebellar pathology,             ry antibody generated against calbindin D-28k (CB) 
                    thus in the present study the localisation and ap-            (Sigma) at dilution of 1 : 200. As the secondary an-
                    pearance of calbindin expressing cells in different           tibody anti-rabbit IgG was used. For visualization of 
                    developmental stages and pathological conditions              the reaction sites the sections were treated with pe-
                    occurring at the time of cerebellar maturation were           roxidase (brown stain) or alkaline phosphatase (red 
                    examined.                                                     stain). Some sections were counterstained by tolu-
                                                                                  idin blue.
                    Material and Methods
                       The investigations were carried out on 33 human            Results
                    brains  obtained  following  autopsy.  The  cerebellar            In the normal cerebellar cortex of newborns all 
                    cortex of cases with paraneoplastic cerebellar dege-          Purkinje cells displayed intense calbindin immuno-
                    neration, cerebellar neuronal migration disturbances          reactivity (Fig. 1).Their cell bodies were strongly sta-
                    and uninjured controls from the age of 30 weeks of            ined and their processes were also positive. Few cells 
                    gestation to 2 years were examined.                           scattered in the external granule layer were immu-
                       Nine brains of children who died in the course of          noreactive. The intensity of staining increased with 
                    neoplastic diseases, at age 2 months-2 years have             the development of Purkinje cells soma. The dendri-
                    been studied. The brain was involved neither by tu-           tic tree arborisation in the molecular layer was well 
                    mour nor cerebral and meningeal metastases. The               visible. In the older cases dendrites of Purkinje cells 
                    neoplasms had various localisations. Autopsy exami-           exhibited high calbindin immunoreactivity imparting 
                    nation showed 5 neuroblastomas, hepatoblastoma,               an intense coloration to the molecular layer where 
                    adenocarcinoma of submandibular gland, and 2 ca-              they arborize. 
                    ses of histiocytosis. Seven of them were not treated;             The Purkinje cells located in cerebellar heteroto-
                    others were treated by polytherapy (chemotherapy              pias and dysgenesias were morphologically well de-
                    and radiotherapy). Antimitotic drugs were used in             veloped and strongly immunostained with CB antibo-
                    classical compositions.                                       dy (Fig. 2). The disorderly scattered cell bodies with 
                       Ten brains with abnormal clusters of neurons ar-           well visible dendritic arborisation were calbindin im-
                    rested in the cerebellar white matter and dysgene-            munopositive. The intensity of calbindin immunore-
                    sias were chosen to the study. The age of cases was           activity corresponded to age matched controls.
                    Folia Neuropathologica 2009; 47/4                                                                                     301
                  Milena Laure-Kamionowska, Danuta Maślińska
                     Fig. 1A-D. Calbindin immunoreactivity in the Purkinje cells of normal cerebral cortex. A. Premature newborn 
                     30 weeks of gestation. Arrow: immunoreactive cells in the external granular layer. Insert: Purkinje cell soma. 
                     Magn. ×20, insert ×40; B. Premature newborn 32 gestational weeks; C. Newborn 36 gestational weeks; D. 
                     Full term newborn; Magn. B,C,D ×40.
                     Fig. 2A-C. Calbindin immunoreactivity in the heterotopic cells in the cerebellar white matter. A. CalbindinD28K 
                     positive cells scattered in the white matter. Full term newborn. Magn. 20×; B. Well stained calbindin positive 
                     soma of the heterotopic cells, the same case. Magn. 40×; C. Group of heterotopic cells with well visible arbori-
                     sation. Two months old infant. Magn. 20×, insert: cortical Purkinje cell, age matched control, Magn. ×20.
                     In paraneoplastic cerebellar degeneration a dif-     emerged. The intensity of calbindin immunoreactivi-
                  fuse loss of Purkinje cells was observed (Fig. 3). In   ty decreased. The neurons changed their shape and 
                  the cells undergoing degeneration the progressive       formed cell globose spheroids. Segmentally unsha-
                  decrease of calbindin was found. Among the Pur-         pely formed cell shadows were seen. The progressive 
                  kinje neurons some steps of calbindin soma disinte-     decrease of calbindin immunoreactivity beside cells 
                  gration were determined. Beside normal appearing        soma was observed in the dendrites. The dendrites 
                  neurons  the  cells  with  fade,  dissolute  cytoplasm  became shortened and tortuous. Dendritic tree disin-
                  302                                                                                 Folia Neuropathologica 2009; 47/4
                                                                                                    Calbindin neurons in human pathology
                      Fig. 3A-F. Calbindin immunoreactivity in the paraneoplastic cerebellar degeneration. A. Diffuse loss of Purkinje 
                      cells, 6 months old infant, Magn. ×4; B. For comparison calbindin positive cells in the normal age matched 
                      cerebellum, Magn. ×4; C. The progressive decrease of calbindin immunoreactivity, one year old infant, Magn. 
                      ×20; D. Advanced disintegration of Purkinje cells, globoid shape of the preserved cells, Magn. ×20; E. Different 
                      stages of disintegration of Purkinje cells, Magn. ×40; F. Preserved fragments of dendritic tree, loss of Purkinje 
                      cell soma, Magn. ×40.
                  tegration led to loss of its continuity. The molecular    -D28k immunoreactive cells are observed in the ven-
                  layer was gradually devoid of calbindin positive den-     tricular zone when neurones become ready to start 
                  drites. However in several parts of cerebellar cortex     migration and differentiation. Parvalbumin is expres-
                  the fragmented dendritic tree in the molecular layer      sed later in development. Some of calcium binding 
                  was preserved, while the Purkinje cell soma comple-       proteins like calretinin are transiently expressed in 
                  tely disappeared.                                         specific cellular subpopulations. It has been postu-
                                                                            lated that calcium binding proteins are important in 
                  Discussion                                                the control of cell division, process outgrowth and 
                                                                            cell movement since all these activities are closely 
                      During normal development of the human cere-          related  to  the  intracellular  calcium  concentration 
                  bellum the cell specific distribution of calcium bin-     [2]. Different calcium binding proteins may function 
                  ding proteins (CABPs) is found [3,10,14]. Calbindin       differently at different developmental stages [6]. In 
                  (CB) appears early, at 4-5 gestational week calbindin     the third trimester of gestation calbindin-D28k im-
                  Folia Neuropathologica 2009; 47/4                                                                           303