Defining the borders of cortical tubers
To discriminate between perituberal cortex and tubers, it is first critical to demarcate the cortical tubers themselves in histological sections. Based on immunohistochemical analysis, we have found that astrogliosis is the best criterion for morphological discrimination between tubers and the surrounding neocortical parenchyma (Figure 1). In addition to providing a clear histological delineation of tubers, this proposal is in line with MRI data where both T1- and T2-weighted images correlate with the level of tissue sclerosis/gliosis [13]. Several markers of astrocytes were used to determine tuber astrogliosis and accordingly tuber borders. These included markers typical for gray matter protoplasmic astrocytes such as glutamine synthetase (GS) and the astrocyte specific glutamate transporters (EAAT1 and EAAT2), all of which showed profoundly lower levels in tubers (Figure 1). In addition, markers that are seen in reactive/gliotic astrocytes such as glial fibrillary acidic protein (GFAP), vimentin, CD44, αB-Crystallin, and S100 revealed striking increases in tuber tissue (Figure 1, only some shown). Tuber borders were thus clearly outlined, segregating perituberal protoplasmic astrocytes from tuber gliosis (Figure 1). We observed that the borders of tubers were not always smooth, displaying uneven profiles with many peripheral protrusions (Figure 1a,b,c,e). Long processes of tuber astrocytes penetrated into the perituberal parenchyma and intermingled with neighboring protoplasmic astrocytes (Figure 1f). Furthermore, small foci of astrogliosis (microtubers, see below) were regularly found near the tuber borders (Figure 1a,b,c).
Tuber-specific cellular abnormalities in perituberal MRI-normal tissue
Most of the observations presented in this section were obtained from MRI-normal, epileptogenic perituberal tissue (n = 10) that was dissected during surgery separately from tubers. Macroscopic tubers were never found in these samples of tissue.
Microtubers are common pathological feature of perituberal tissue
In contrast to the highly gliotic tubers, the perituberal gray matter was populated mainly with protoplasmic astrocytes displaying high levels of GS and low levels of GFAP (Figure 2a,b). To detect abnormal cells typical for tubers within perituberal tissue, we used several markers specific for neurons and glial cells and known to be increased in giant cells and cytomegalic neurons [14,15], as well as p-S6, to look for mTOR activation [16]. In every section from every specimen of perituberal tissue, we found aberrant cells characteristic of tubers: giant cells, gliotic astrocytes, and cytomegalic neurons.
In the perituberal gray matter, giant cells were usually surrounded by fibrous-like astrocytes with long processes displaying high levels of GFAP and CD44. These small astrogliotic islands were clearly outlined from the neighboring normal parenchyma containing protoplasmic astrocytes (Figure 1a,b,c; 2). We previously designated these microscopic islands as “microtubers” [11], in contrast to the canonical cortical “macrotubers” detected by MRI. They were relatively homogeneous in size, with an average diameter of 284.7 ± 17.3 μm (n = 70; range: max 461.6 μm, min 158.6 μm). If we consider the shape of microtubers as roughly spherical, we estimate that one microtuber is composed, on average, of ~ 27 astrocytes and includes ~ 20 neurons.
Two types of microtubers were distinguished based on the shapes and immune profile of astrocytes. The majority (∼80%) of microtubers (which we designated as type I) contained many astrocytes with long processes that radiated for several hundred micrometers into the adjacent gray matter, which was occupied by protoplasmic astrocytes (Figure 2a,b,d). The processes of these cells were devoid of the miniature lamellipodial-like processes that are a characteristic feature of protoplasmic astrocytes. Such structural feature was especially obvious when cells were immunostained for the plasma membrane glycoprotein CD44 (Figure 2b1). A minority (~20%) of microtubers (designated type II) were largely composed of astrocytes with processes of normal length endowed with many miniature lamellipodial leaf-like extensions that produced the typical bushy-like appearance of protoplasmic astrocytes (Figure 2a,c). However, in contrast to typical protoplasmic astrocytes, these cells were CD44+ (Figure 2c). It is worth noting that some type II microtubers contained only a few (2–4 in a plane of inspection) reactive-like astrocytes neighboring a giant cell (Figure 2e).
We suggest that the astrocytes with long, non-branched processes in type I microtubers are similar in many ways to the CD44+ long-process/interlaminar astrocytes in gray matter and/or to fibrous astrocytes in white matter, whereas astrocytes with processes of protoplasmic astrocytes size and shape, but CD44+, in type II microtubers are reactive protoplasmic astrocytes. To test this hypothesis we used immunostaining for SPARC/osteonectin, a glycoprotein we have found to be a characteristic marker of CD44+ interlaminar and fibrous astrocytes in human brain [17]. Indeed, type I microtubers contained many SPARC+ astrocytes whereas only a few SPARC+ cells were observed in type II microtubers (11.8 ± 0.824 per microtuber in type I vs 0.824 ± 0.3 in type II, p < 0.001) (Figure 3a,b). In addition, all SPARC+ astrocytes were CD44+ and had clearly outlined, long main branches without lamellipodial-like processes (Figure 3c). It should be noted that many giant cells also showed immunolabelling for SPARC (Figures 3b,d; 4c,d).
Astrocytes in both types of microtubers were characterized by diminished immunoreactivity for glutamate transporters (EAAT2 and EAAT1) (only EAAT2 was quantified, OD, arbitrary units, 0.1 ± 0.03 in microtubers vs 0.6 ± 0.3 in surrounding normal parenchyma, p < 0.001) and for GS (OD, arbitrary units, 1199.7 ± 296.6 in microtubersvs 9874.4 ± 1321.8 in surrounding normal parenchyma, p < 0.001) (Figure 3e-g). This decrease in GS and glutamate transporters was particularly prominent in the central part of microtubers, where many neurons were surrounded by astrocytes lacking these proteins (Figure 3e-g).
Many astrocytes in microtubers showed p-S6 immunoreactivity (Figure 4a,c,d). Especially prominent p-S6 expression was observed in microtubers located in deep cortical layers (255 of 349 astrocytes in V-VI layers vs. 46 of 123 astrocytes in upper I-IV layers, p < 0.001).
To gain additional insight into the characteristics of astrocytes in microtubers, we tested activation (phosphorylation) of p44/42 MAPK (p44), which was previously shown to be activated in tubers [18]. We found that many astrocytes in microtubers displayed a high level of p44 (Figure 4b,c). Microtubers located in deep cortical layers and in white matter contained more p44+ astrocytes than those in upper gray matter layers (56 of 121 cells in upper layers vs. 156/184 in deep layers, p < 0.001). Only a few (6/61) giant cells revealed immunoreactivity for p44 (Figure 4c). Many p44+ astrocytes were also immunopositive for p-S6 (in deep cortical microtubers ~ 80%, 214 of 305 p44+ astrocytes) (Figure 4c). Many SPARC+ astrocytes were also p-S6+ (Figure 4d).
The appearance of reactive-like astrocytes in the immediate vicinity of giant cells in microtubers raised the possibility that giant cells might modulate their environment. We examined microtubers for microglial reaction as a marker of inflammatory brain parenchymal disturbances. In 45/47 microtubers tested with CD68 and LN-3 immunostaining, there was no prominent activation of microglia detected (shown only CD68, Figure 5a). Optical density of CD68 immunolabelling did not differ in microtubers from surrounding normal parenchyma (6150 ± 885 arbitrary units per 1 mm2 in microtubers vs 6030 ± 799 in normal parenchyma, P = 0.922). In only 2/47 microtubers, activated microglia were observed in large areas that included the microtubers but extended far beyond their boundaries (not shown). We also used immunostaining for Iba1, as a general marker for both resting and activated microglia, and did not find significant microglial changes in microtubers (Figure 5b). Numbers of Iba1+ microglial cells did not differ significantly from surrounding normal parenchyma (61.3 ± 7.66 cell per 1 mm2 in microtubers vs 55.5 ± 8.08 in normal parenchyma, P = 0.62).
We assayed also whether giant cells contain tumor necrosis factor alpha (TNFα), as shown previously [18]. TNFα is a cytokine that is responsible for induction of reactive changes in astrocytes in variable brain pathologies [19]. We found that many giant cells were TNFα+ (in microtubers 14 of 21 cells, in macrotubers 77/115, p = 0.722) (Figure 5c,d).
In perituberal tissue, in addition to giant cells in microtubers, we also found scattered giant cells surrounded by normal appearing protoplasmic astrocytes (Figure 5e,f,g). There were <1/10th as many of these isolated giant cells in comparison to giant cells within microtubers (24 versus 235 cells), and they were found predominantly in upper (I – III) cortical layers (19 of 24).
Giant cells in the perituberal gray matter (both in microtubers and isolated) never reached as large a size as those within tubers (374.3 ± 22.2 μm2 (n = 20) vs 888.9 ± 40.88 μm2 (n = 20), respectively, p < 0.001) and never had as long and thick processes as giant cells in tubers (compare those in Figure 5e-g with those in Figure 6a-c). We also did not find giant cells immunopositive for p75 neurotrophin receptor (p75) in perituberal tissue although p75+ giant cells were often observed in tubers (106/178 in tubers vs. 0/34 in microtubers, p < 0.001) (Figure 6c). Such differences in giant cells might indicate different developmental routes or different growth and ‘maturation’ of giant cells.
An examination of a single TSC patient’s hippocampus that had a normal MRI appearance but was resected as part of the epileptogenic zone revealed microtubers and cytomegalic neurons in Ammon’s horn (Figure 6d,e), as well as in the hilus (not shown). This further supports the idea that microtubers represent important parts of epileptogenic zones apart from tubers.
Cytomegalic neurons in perituberal tissue
Analysis of specimens stained with the pan-neuronal marker NeuN did not show any peculiar features of the neuronal population (cellular density, cytoarchitecture, aberrant cellular types) in microtubers (Figure 6f).
Cytomegalic/dysplastic neurons similar to those characteristically found in tubers were also consistently found in perituberal gray matter. These cells were morphologically identified as large cells with long processes that were immunopositive for neuronal (neurofilaments [NF], microtubule-associated proteins [MAP2], and NeuN) markers and immunonegative for giant cell markers (GS, vimentin, nestin) (Figure 6g,h). Many giant cells in perituberal tissue and tubers were also positive for NF and/or MAP2 (Figure 6i,j, shown only NF). However, in contrast to cytomegalic neurons, the NF+/MAP+ giant cells expressed astrocyte-specific markers, e.g. GS and vimentin, had a bizarre shape, and one or several, eccentrically-located nuclei.
The majority of cytomegalic (67 of 73), as well as some normal-shaped neurons, in perituberal tissue were p-S6 immunoreactive (Figure 6h). However, we did not consider this staining property to be a reliable marker of a neuronal abnormality, since many neurons in neocortical and hippocampal gray matter in samples of brains obtained from temporal lobe epilepsy and other non-epilepsy surgeries revealed high levels of p-S6 immunoreactivity (unpublished data).
It is important to note that cytomegalic neurons were not a part of perituberal microtubers (found in 0/31 microtubers examined by double staining for NF/MAP2 and GFAP). In addition, we did not observe single giant cells near cytomegalic neurons (12 giant cells and 14 cytomegalic neurons examined in double immunostains for NF/MAP2 and p-S6). We also did not detect any significant alterations in NG2 cells or oligodendrocytephenotypes in microtubers (not shown), similar to our previous analysis of tubers [20].
Astrocyte heterogeneity in cortical tubers
As emphasized above, one of the most prominent features of tubers was a high level of astrogliosis. In 7/16 tubers, severe astrogliosis completely occupied the tuber gray matter (to pia surface) and almost every astrocyte was devoid of GS and glutamate transporter (EAAT1 and EAAT2) immunoreactivity. In the other 9/16 tubers, we found that upper cortical layers (usually layer 1 and 2) were populated with protoplasmic astrocytes and many microtubers were located near the tubers (Figure 1a,b,c). Small “islands” of protoplasmic astrocytes were observed within tuber highly gliotic tissue that produced a patchy pattern due to intermingled gliotic and non-gliotic areas (Figure 1e, 7a). Because we did not make 3-D reconstructions of the whole tubers we cannot rule out the idea that these island-like areas of normal brain parenchyma were not in reality deep invaginations of perituberal, normal parenchyma.
Although most tubers were composed of a homogeneous population of gliotic astrocytes, in some areas within tubers it was possible to discriminate groups of astrocytes displaying higher levels of vimentin, p-S6, and p44 than neighboring gliotic astrocytes (Figure 7b,c,d). These cells were grouped around giant cells and might correspond to microtubers with astrocytes that did not reach the “final stage of gliotic maturation” observed in neighboring highly gliotic tuber astrocytes. The average diameter of such groups was 329 ± 15.1 μm (n = 15; range: max 459.3 μm, min 236.7 μm), overlapping extensively with the dimensions of the perituberal microtubers. Indeed, these groups within tubers exceeded the average dimension of microtubers in the perituberal parenchyma by less than the size of one astrocyte domain (~100 μm, not including the area of overlapping processes from neighboring cells [21]).
The phenotype of astrocytes located at the tuber border zones was different from that of astrocytes in the central part of the tubers, as well as from that of normal protoplasmic astrocytes in the neighboring gray matter. These ‘transitional’ areas, which we speculate represent developing gliosis in tubers, were populated with astrocytes that had characteristic features consistent with ongoing astrocyte changes. 1) Only in these areas were there unusual astrocytes with shapes of fibrous astrocytes (cells with long processes devoid of miniature leaf-like protrusions) and yet with high levels of GS, EAAT1, and EAAT2, which are characteristic features of protoplasmic astrocytes (Figure 7e-h). Taking into consideration that the majority of these unusual astrocytes did not show immunoreactivity for SPARC (Figure 7f,h), we considered them to be astrocytes with mixed protoplasmic and fibrous features. 2) Many astrocytes in these areas were immunopositive for phosphorylated p44 MAPK. These p44+ astrocytes showed minimal immunoreactivity for GS and glutamate transporters. (Figure 8a-c). 3) p-S6+ astrocytes predominated in these areas and often immunoreactivity for p-S6 colocalized with p44 and SPARC (Figure 8b,d). Both p44+ and p-S6+ astrocytes expressed SPARC and revealed low levels of GS and glutamate transporters, whereas astrocytes with high levels of GS and glutamate transporters were SPARC immunonegative (Figure 7f,h; 8c,d). Thus, many astrocytes in these “transition zones” display features of both protoplasmic and long-process astrocytes. These may well reflect a phenotype in transition.
The level of microglial activation (based on immunoreactivity for CD68 and Iba1) in these transitional areas was similar to that in tubers and significantly exceeded that of the non-gliotic areas surrounding tubers (not shown).