Detection of amyloid beta aggregates in the brain of BALB/c mice after Chlamydia pneumoniae infection Neuroinflammation , initiated by cerebral infection , is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer 's disease -LRB- AD -RRB- . We investigated whether Chlamydia pneumoniae -LRB- Cpn -RRB- infection results in extracellular aggregation of amyloid beta -LRB- Aβ -RRB- in BALB/c mice . At 1 week post intranasal infection -LRB- p.i. -RRB- , Cpn DNA was detected predominantly in the olfactory bulbs by PCR , whereas brains at 1 and 3 months p.i. were Cpn negative . At 1 and 3 months p.i. , extracellular Aβ immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected . However , these extracellular Aβ aggregates showed morphological differences compared to extracellular Aβ aggregates detected in the brain of transgenic APP751SL/PS1M146L mice . These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like Aβ plaques in the brain of BALB/c mice , as suggested before . However , future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis . Introduction The most common cause of dementia is Alzheimer 's disease -LRB- AD -RRB- , which manifests in a familial and a sporadic form . The neuropathology in both conditions is characterized by neuritic or senile plaques , consisting mainly of amyloid beta protein -LRB- Aβ -RRB- and neurofibrillary tangles with hyperphosphorylated tau protein as its key components . Mutations in the amyloid precursor protein -LRB- APP -RRB- , presenilin-1 -LRB- PS-1 -RRB- or -2 -LRB- PS-2 -RRB- genes have been associated with the familial form of AD -LSB- 27 -RSB- . However , the most common type of this disease , sporadic AD , is not due to these mutations , and although risk factors have been identified , the initiating trigger is still unknown -LSB- 18 -RSB- . The potential importance of infections in the development of sporadic AD has been recently recognized -LSB- 1 , 21 , 31 -RSB- . Specifically , not only various viruses like the human immunodeficiency virus , herpes simplex virus and cytomegalovirus but also bacteria like Borrelia burgdorferi , have been linked to the process of neurodegeneration -LSB- 6 , 12 , 13 , 17 , 20 -RSB- . Some data also suggest that Chlamydia pneumoniae -LRB- Cpn -RRB- should be added to the list of pathogens possibly involved in the pathogenesis of AD . Cpn is a widespread , obligatory intracellular pathogen , often causing acute respiratory infections including pneumonia , sinusitis and bronchitis -LSB- 10 , 11 , 15 -RSB- . This microorganism has also been implicated in a variety of severe , chronic diseases including some disorders of the central nervous system -LSB- 7 , 14 , 30 , 32 -RSB- . In an initial study , Balin et al. demonstrated the presence of Cpn in brains of 17 out of 19 patients with sporadic AD , using a variety of techniques , including PCR , electron microscopy and immunohistochemistry , while the pathogen could only be demonstrated in 1 out of 19 controls -LSB- 2 -RSB- . The presence of Cpn was demonstrated in various cell types like microglia , astrocytes , perivascular macrophages and monocytes , and predominantly in brain areas linked with AD pathology . Recently , these observations were confirmed -LSB- 8 -RSB- . On the contrary , others were unable to detect Cpn in AD patient tissues -LSB- 9 , 22 , 23 , 28 -RSB- . The only animal study so far supporting a role for this pathogen in AD demonstrated an AD-like pathology in the brain of BALB/c mice post Cpn infection -LSB- 19 -RSB- . Three-month-old mice were inoculated intranasally with Cpn and the authors claimed that this resulted in the deposition of Aβ in the brain up to 3 months post infection with increasing pathology as the infection progressed . Based on these results , it was concluded that in naïve BALB/c mice , AD-like pathology could be induced by Cpn infection . In spite of these results , doubts remain concerning the amyloid-like plaques as being only unrelated elevations in amyloid -LSB- 24 -RSB- . Because of the possible implications of this study and the need for confirmation , it was our aim to repeat these experiments under quite corresponding conditions . Moreover , we compared our findings with the pathology found in a transgenic mouse model of AD . Materials and methods Chlamydia pneumoniae The Cpn strain , TWAR 2043 -LRB- ATCC -RRB- was cultured and continuously propagated on Hep2 cells as previously described -LSB- 25 -RSB- . Bacterial titres were determined by titration in Hep2 cells -LSB- 7 -RSB- and staining with a monoclonal Chlamydia LPS-specific antibody followed by a FITC-conjugated secondary antibody -LRB- RR-402 ; DAKO , Glostrup , Denmark -RRB- . Titres were expressed as the number of inclusion forming units -LRB- IFU -RRB- per ml . Animals and infection protocol A total of 43 female , specific pathogen-free inbred BALB/c mice were obtained from Charles River -LRB- Someren , The Netherlands -RRB- . Animals were kept under standard housing and care conditions at the Central Animal Facilities of Maastricht University -LRB- Maastricht , The Netherlands -RRB- . All experimental procedures were approved by the Ethical Committee for the Use of Experimental Animals of Maastricht University . At 3 months of age n = 24 mice were inoculated intranasally with 107 IFU Cpn in 10 μl -LRB- 5 μl per nostril -RRB- , and were analyzed at 1 week , 1 month or 3 months p.i. , respectively , as shown in Table 1 . Pilot experiments revealed that this dosage ensures both survival of the mice and optimal dissemination throughout the body . Another n = 15 mice were mock infected with a sucrose-phosphate-glucose solution -LRB- SPG , solution for storage of Cpn -RRB- at 3 months of age and were also analyzed at 1 week , 1 month or 3 months after mock infection , as shown in Table 1 . In addition , two mice , which did not receive any treatment , were investigated at 4 months of age , as well as two mice at 6 months of age -LRB- Table 1 -RRB- . Table 1Numbers of animals per group investigated in the present studyMouse strain ; analysisInfection with CpnMock infectionNo treatment1w1m3m1w1m3mM4M6M17BALB/c ; PCR444111 -- -- -- BALB/c ; IHC -- 66 -- 6622 -- APP751SL/PS1M146L -- -- -- -- -- -- -- -- 1CpnChlamydia pneumoniae , IHC immunohistochemistry , 1w/1m/3m , analysis 1 week -LRB- or 1 month or 3 months , respectively -RRB- after Cpn infection or mock infection at 3 months of age . M4/M6/M17 , 4 , 6 or 17 months of age , respectively In addition , a 17-month-old transgenic mouse expressing human mutant APP751 -LRB- carrying the Swedish and London mutations KM670/671NL and V717I , under the Thy1 promoter -RRB- and human mutant presenilin-1 -LRB- PS-1 M146L , under the HMG promoter -RRB- was used -LRB- APP751SL/PS1M146L mouse ; for a detailed description see refs -LSB- 4 -RSB- and -LSB- 24 -RSB- -RRB- -LRB- Table 1 -RRB- . This APP751SL/PS1M146L mouse was bred on a CBA -LRB- 12.5 % -RRB- × C57Bl6 -LRB- 87.5 % -RRB- background . Detection of Cpn DNA in brain tissue with real-time PCR As shown in Table 1 , 15 mice were used to detect Cpn DNA in brain tissue with real-time PCR at different time points after infection with Cpn or mock infection . To this end the animals received a weight-adjusted dose of pentobarbital -LRB- Nembutal ; Sanofi Sante B.V. , Maassluis , The Netherlands -RRB- and were then perfused with sterile phosphate buffered saline . Brains were removed quickly and dissected into five different regions , i.e. , olfactory bulbs , cerebellum , hippocampus , subventricular zone and neocortex . The isolated regions were snap-frozen in liquid nitrogen and stored at − 80 °C until further processing . DNA was isolated from brain material using methods previously described -LSB- 5 -RSB- . The Nanodrop ® ND-1000 , a full-spectrum spectrophotometer -LRB- 220 -- 750 nm -RRB- was used to calculate the DNA content of each sample . Finally , the Cpn DNA copy number/μg DNA in the collected brains was determined by real-time PCR -LRB- ABI Prism 7000 ; Applied Biosystems , Foster City , CA , USA -RRB- using the following primers and probe ; forward -LRB- 5 ′ TCCGCATTGCTCAGCC3 ′ -RRB- , reverse -LRB- 5 ′ AAACAATTTGCATGAAGTCTGAGAA3 ′ -RRB- and probe -LRB- 5 ′ - FAM-TAAACTTAACTGCATGGAACCCTTCTTTACTAGG3 ′ - TAMRA -RRB- as described previously -LSB- 5 , 29 -RSB- . All PCR runs were performed in duplo . Mean Cpn DNA copy numbers/μg brain DNA of three independent experiments -LRB- ± SEM -RRB- was calculated . Then , comparisons between all groups of mice , analyzed for the presence of Cpn DNA in brain tissue , were performed with three-way ANOVA with Treatment -LRB- Cpn infection or mock infection -RRB- , Time-point of analysis -LRB- 1 week , 1 month or 3 months p.i. -RRB- and Brain region -LRB- olfactory bulbs , cerebellum , hippocampus , subventricular zone or neocortex -RRB- as fixed factors . P values smaller than 0.05 were considered statistically significant . Calculations were performed with SPSS v. 12.0.1 for Windows -LRB- SPSS , Chicago , IL , USA -RRB- . Detection of Aß aggregates in brain tissue with immunohistochemistry As also shown in Table 1 , n = 29 mice were used to detect Aβ aggregates in brain tissue with immunohistochemistry at different time points after infection with Cpn , mock infection or without any treatment , respectively . To this end , the animals received a weight-adjusted dose of pentobarbital -LRB- Nembutal ; Sanofi Sante B.V. , Maassluis , The Netherlands -RRB- and were then subjected to intracardial perfusion fixation , as previously described -LSB- 26 -RSB- . In case of the APP751SL/PS1M146L mouse , anaesthesia was performed with chloral hydrate -LRB- 10 % aqueous solution , 0.005 ml/g body weight , i.p. -RRB- . Brains were removed rapidly and post-fixed for 2 h at 4 °C in the same fixative , omitting the glutaraldehyde . In case of the APP751SL/PS1M146L mouse , the brains were halved in the midsagittal line . Brain tissues were then cryoprotected by immersion in 30 % sucrose in Tris-buffered saline at 4 °C overnight . Afterwards , brains -LRB- or brain halves , respectively -RRB- were quickly frozen and stored at − 80 °C until further processing . For immunohistochemical analysis , the entire brains -LRB- or the right brain half in case of the APP751SL/PS1M146L mouse -RRB- were cut into entire series of 30-μm-thick frontal sections on a cryostat -LRB- Leica CM 3050 S ; Leica , Nussloch , Germany -RRB- . After cutting , sections were again stored at − 80 °C until further processing . Immunohistochemical detection of Aβ -LRB- as well as of glial fibrillary acidic protein -RRB- was performed with standard immunofluorescence procedures using a first layer , consisting of a combination of mouse anti-human amyloid 17 -- 24 -LRB- 4G8 clone ; 1:1,200 in TBS-T ; Sigma-Aldrich , St Louis , USA -RRB- -LSB- 3 , 16 , 33 -RSB- and polyclonal rabbit anti-mouse GFAP -LRB- 1:1,600 in TBS-T ; DAKO , Glostrup , Denmark -RRB- . The corresponding secondary antibodies were donkey anti-mouse Alexa 488 -LRB- 1:100 in TBS-T ; Molecular Probes -RRB- and donkey anti-rabbit Alexa 594 -LRB- 1:100 in TBS-T ; Molecular Probes -RRB- . Finally , counterstaining was performed with Hoechst -LRB- 1:500 in TBS ; Sigma , St Louis , MO , USA -RRB- and sections were mounted on gelatine-coated glasses . As a negative control , sections were only incubated with the secondary antibodies . Detection of extracellular Aß aggregates in brain tissue with thioflavin-S For the detection of extracellular , fibrillary Aβ , a standard thioflavin-S -LRB- Sigma -RRB- staining was performed . Finally , the sections were counterstained with Hoechst and mounted as described above . Microscopy and photography Microscopic inspection of sections was performed with a MBF Bioscience Stereo Investigator Confocal Spinning Disk -LRB- SI-SD -RRB- system -LRB- MBF Bioscience ; Williston , VT , USA -RRB- , consisting of a modified Olympus BX51 fluorescence microscope -LRB- Olympus , Tokyo , Japan -RRB- with UPlanSApo objectives 10 × -LRB- N.A. = 0.4 -RRB- , 40 × -LRB- N.A = 0.9 -RRB- and 100 × -LRB- oil ; N.A = 1.4 -RRB- ; Olympus , customized spinning disk unit -LRB- DSU ; Olympus -RRB- , computer-controlled excitation and emission filter wheels -LRB- Olympus -RRB- , three-axis high-accuracy computer-controlled stepping motor specimen stage -LRB- 4 × 4 Grid Encoded Stage ; Ludl Electronic Products , Hawthorne , NY , USA -RRB- , linear z-axis position encoder -LRB- Ludl -RRB- , ultra-high sensitivity monochrome electron multiplier CCD camera -LRB- 1,000 × 1,000 pixels , C9100-02 ; Hamamatsu Photonics , Hamamatsu City , Japan -RRB- and controlling software -LRB- MBF Bioscience -RRB- . In case of sections processed for the detection of Aß and GFAP , digital RGB image stacks showing the same microscopic field in 50 consecutive focal planes with a distance of 0.5 μm between the focal planes were generated with the 40 × objective . In the case of sections stained with thioflavin-S , digital image stacks showing the same microscopic field at 150 consecutive RGB focal planes with a distance of 0.2 μm between the focal planes were generated with the 100 × objective . From these image stacks three-dimensional reconstructions -LRB- maximum intensity projections -RRB- were produced with Imaris software -LRB- Version 4.0 ; Bitplane , Zurich , Switzerland -RRB- . In addition , low-power photomicrographs -LRB- 10 × -RRB- were taken from sections stained with thioflavin-S . Final figures were constructed using Corel Photo-Paint v. 11 and Corel Draw v. 11 -LRB- Corel , Ottawa , Canada -RRB- . Only minor adjustments of contrast and brightness were made , without altering the appearance of the original materials . No deconvolution was performed on images obtained by confocal microscopy . Results Presence of Cpn in the brain of BALB/c mice infected with Cpn At 1 week p.i. , the mean Cpn DNA copy number per μg DNA was 310.97 ± 61.6 -LRB- mean ± SEM -RRB- in the olfactory bulbs , 2.6 ± 0.8 in the cerebellum and 7.9 ± 0.8 in the hippocampus , while no Cpn DNA was detected in the subventricular zone and the neocortex -LRB- Table 2 -RRB- . No Cpn DNA was detected in any brain region of the Cpn infected mice at 1 and 3 months p.i. . Furthermore , Cpn DNA was not detected in the brain of mock-infected BALB/c mice at any time point . Statistical analysis revealed significant differences among the groups of mice with respect to Treatment -LRB- T -RRB- -LRB- P = 0.024 -RRB- , Time point of analysis -LRB- Tpoa -RRB- -LRB- P = 0.008 -RRB- and Brain region -LRB- Br -RRB- -LRB- P = 0.002 -RRB- , as well as with respect to the combinations T -- Tpoa -LRB- P = 0.008 -RRB- , T -- Br -LRB- P = 0.002 -RRB- , Tpoa -- Br -LRB- P < 0.001 -RRB- and T -- Tpoa -- Br -LRB- P < 0.001 -RRB- . Table 2Detection of Cpn DNA in various brain regions -LRB- olfactory bulbs , cerebellum , hippocampus , subventricular zone and neocortex -RRB- with real-time PCR at 1 week , 1 month and 3 months post infection1 week p.i. 1 month p.i. 3 months p.i.Olfactory bulbs310 .97 ± 61.600 Cerebellum2 .6 ± 0.800 Hippocampus7 .9 ± 0.800 Subventricular zone000Neocortex000Data were expressed as mean values of three independent experiments ± SEM Detection of Aβ and GFAP in the brain of BALB/c and APP751SL/PS1M146L mice Amyloid beta immunoreactivity was found in the brains of all mice , i.e. in Cpn-infected BALB/c mice at both 1 and 3 months p.i. -LRB- Fig. 1a -RRB- , mock-infected BALB/c mice at both 1 and 3 months after mock infection -LRB- Fig. 1b -RRB- , not inoculated BALB/c mice -LRB- Fig. 1c -RRB- and in APP751SL/PS1M146L mice -LRB- Fig. 1d -RRB- . However , Aβ-immunopositive aggregates were found only occasionally in the brains of the BALB/c mice -LRB- on average one to two aggregates per section without a preference for a certain brain region -RRB- , whereas they were found abundantly in hippocampus and neocortex of APP751SL/PS1M146L mice -LRB- see also Fig. 2H in -LSB- 24 -RSB- -RRB- . Furthermore , there were several qualitative differences between the APP751SL/PS1M146L and the BALB/c mice . First , in the brain of APP751SL/PS1M146L mice , the materials immunopositive for Aβ formed a dense core , devoid of Hoechst-positive structures -LRB- i.e. cell nuclei -RRB- . In contrast , the materials immunopositive for Aβ in the brain of BALB/c mice did not present with a dense core , and Hoechst-positive structures were scattered within these materials . Second , the astrocytic reaction in the brain of APP751SL/PS1M146L mice , surrounding the Aβ-immunopositive materials , was much stronger than in the brain of BALB/c mice -LRB- arrowheads in Fig. 1a -- d -RRB- . Third , in the brain of APP751SL/PS1M146L mice , cells with intracellular deposits of Aβ were found -LRB- asterisk in Fig. 1d -RRB- , which was not observed in the brain of BALB/c mice , irrespective of treatment . Fig. 1Immunohistochemical detection of Aβ -LRB- green -RRB- and GFAP -LRB- red -RRB- in 30 μm-thick cryostat sections counterstained with Hoechst -LRB- blue -RRB- from the brain of a 6-month-old Cpn-infected BALB/c mouse 3 months post infection -LRB- a -RRB- , a 6-month-old mock infected BALB/c mouse 3 months after mock infection -LRB- b -RRB- , a 6-month-old BALB/c mouse neither infected with Cpn nor mock infected -LRB- c -RRB- and a 17-month-old APP751SL/PS1M146L transgenic mouse -LRB- d -RRB- . Note that material immunopositive for Aβ was found in the brain of all mice -LRB- arrows -RRB- . However , there were differences between the APP751SL/PS1M146L mouse and the BALB/c mice : -LRB- 1 -RRB- in the brain of the APP751SL/PS1M146L mouse the material immunopositive for Aβ formed a dense core devoid of Hoechst-positive structures -LRB- i.e. cell nuclei -RRB- . In contrast , the material immunopositive for Aβ in the brain of the BALB/c mice did not present with a dense core , and Hoechst-positive structures were scattered within it . -LRB- 2 -RRB- The astrocytic reaction in the brain of the APP751SL/PS1M146L mouse surrounding the material immunopositive for Aβ was much stronger than in the brain of the BALB/c mice -LRB- arrowheads -RRB- . -LRB- 3 -RRB- In the brain of the APP751SL/PS1M146L mouse cells with intracellular deposits of material immunopositive for Aß were found -LRB- asterisk in d -RRB- . This was not observed in the brain of the BALB/c mice . Furthermore , extracellular thioflavin-S-positive structures in the brain of the 17-month-old APP751SL/PS1M146L mouse are shown -LSB- e -LRB- arrowheads -RRB- and f , resembling fibrillary Aβ deposits . Scale bar = 25 μm in a -- d , 100 μm in e and 10 μm in f Detection of thioflavin-S-positive structures in the brain of APP751SL/PS1M146L but not in the brain of BALB/c mice In the brain of APP751SL/PS1M146L mice , extracellular thioflavin-S-positive structures were found -LRB- Fig. 1e , f -RRB- as also previously reported -LSB- 4 , 26 -RSB- , resembling fibrillary Aβ deposits . Such extracellular thioflavin-S-positive structures were not found in the brain of BALB/c mice . Discussion The role of inflammatory factors , predominantly caused by infections , in the initiation or acceleration of neurodegenerative diseases is increasingly recognized . Next to several viruses -LSB- 6 , 12 , 13 , 17 -RSB- , the obligatory intracellular bacterium Cpn has also been linked to a variety of neurodegenerative diseases such as AD . In 1998 , the presence of this pathogen was described in postmortem brains from patients with AD -LSB- 2 -RSB- . That Cpn may contribute to neurodegenerative processes was recently supported by data from our laboratory , as we demonstrated that Cpn is able to infect neurons , ultimately resulting in neuronal cell death in vitro -LSB- 5 -RSB- . However , to the best of our knowledge only one experimental in vivo study has been published so far supporting a role for Cpn in AD -LSB- 19 -RSB- . In this study , naïve BALB/c mice were inoculated intranasally with Cpn at an age of 3 months . Brains were recovered at 1 -- 3 months p.i. , revealing the presence of amyloid-like plaques and astrocyte reactivity . Due to the possible impact of this study , it was our goal to confirm these data by repeating these experiments . Focusing on our results of Cpn-inoculated mice at 3 months p.i. , material immunopositive for Aβ was indeed detected in the brain . Accordingly , at first glance , our results are in line with the study of Little et al. -LSB- 19 -RSB- . Comparing the morphological appearance of this material immunopositive for Aβ with corresponding material in the brain of APP751SL/PS1M146L mice , however , some significant and important differences were observed . First , one characteristic of neuritic -LRB- AD -RRB- plaques is the presence of filamentous , star-shaped aggregates of Aβ fibrils with a thioflavin-S-positive dense core . However , neither such star-shaped aggregates of Aβ fibrils nor extracellular thioflavin-S-positive structures were found in any of the brains of Cpn infected or mock infected BALB/c mice , whereas both lesions were abundant in the brain of APP751SL/PS1M146L mice . Second , no damage was found in the tissue surrounding the material immunopositive for Aβ . Rather Hoechst-positive structures -LRB- i.e. cell nuclei -RRB- could be detected within the material immunopositive for Aβ , a feature that was not seen in the brain of APP751SL/PS1M146L mice . Finally , although astrocytes were found in the vicinity of the material immunopositive for Aβ in the brain of Cpn infected or mock infected BALB/c mice , they were not intimately associated with the Aβ immunoreactivity , as seen in the brain of APP751SL/PS1M146L mice . In summary , except that Aβ plaques were detected in most brains , our data suggest that at 3 months after Cpn infection most characteristic features of AD pathology could not be detected in 6-month-old naïve , non-transgenic BALB/c mice , whereas these were prominent in the brain of the `` control '' APP751SL/PS1M146L mice . It may still be argued that the material immunopositive for Aβ in the brain of the Cpn-infected mice represented early precursors of neuritic plaques . To our surprise , however , nearly identical patterns of Aβ immunoreactivity were detected in mock-infected BALB/c mice as well as in BALB/c mice , neither Cpn infected nor mock infected . This suggests that the observed immunoreactivity for Aβ in the brain of BALB/c mice does not directly result from Cpn infection but might represent unrelated elevations in amyloid . Positive staining for Aβ in the brain of mock-infected mice was also observed by Little et al. -LSB- 19 -RSB- , who found that the amyloid deposition load and size were significantly enhanced in Cpn-infected mice compared to mock infected ones . As outlined above , this was not found in the present study . Furthermore , Little et al. also demonstrated the presence of a small subset of thioflavin-S positive plaques , which were not found in our mice . These discrepancies between the study by Little et al. -LSB- 19 -RSB- and the present study could be due to the fact that Little et al. -LSB- 19 -RSB- used a Cpn strain -LRB- C. pneumoniae , 96-41 , isolated from the postmortem brain of an AD patient -RRB- that was rather different from the Cpn strain used in the present study -LRB- TWAR 2043 , ATCC -RRB- . Notably , Little et al. -LSB- 19 -RSB- , were able to detect Cpn in olfactory tissues of infected mice up to 3 months p.i. , by both light and electron microscopy . In the present study , however , we were only able to detect Cpn DNA in the olfactory bulb of infected BALB/c mice at 1 week p.i. but not at 1 or 3 months p.i. , despite the use of a more sensitive detection method -LRB- real-time PCR -RRB- . As it is well established that the clinical isolates of pathogens are often more infectious than their laboratory counterparts , this could partially explain the discrepancy between both studies . Furthermore , it can not be excluded that differences in solutions used for mock infection -LRB- Hanks balanced salt solution in the study by Little et al. -LSB- 19 -RSB- vs. SPG solution in the present study -RRB- may have affected the final outcome . Yet , in the present study , the immunosignal in mice that were neither Cpn infected nor mock infected appeared somewhat weaker than in the brain of the BALB/c mice that were either Cpn - or mock-infected -LRB- a detailed quantitative analysis was not performed -RRB- . This suggests that intranasal application of Cpn or other infectious agents may trigger the production and deposition of Aβ in the brain of BALB/c mice . For the time being , however , the impact of this remains unclear . In conclusion , our data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD like Aβ plaques in the brain of BALB/c mice , as suggested by Little et al. -LSB- 19 -RSB- . Accordingly , future studies are required to investigate whether Cpn infections may aggravate AD pathology by stimulating the formation of Aβ plaques as has been suggested in atherosclerosis , a disease that is also characterized by pronounced local inflammation .

O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Cellular distribution of vascular endothelial growth factor A -LRB- VEGFA -RRB- and B -LRB- VEGFB -RRB- and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB Members of the vascular endothelial growth factor -LRB- VEGF -RRB- family are key signaling proteins in the induction and regulation of angiogenesis , both during development and in pathological conditions . However , signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells . In the present study , we immunocytochemically investigated the expression and cellular distribution of VEGFA , VEGFB , and their associated receptors -LRB- VEGFR-1 and VEGFR-2 -RRB- in focal cortical dysplasia -LRB- FCD -RRB- type IIB from patients with medically intractable epilepsy . Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity -LRB- IR -RRB- for VEGFA , VEGFB , and VEGF receptors -LRB- VEGFR-1 and VEGFR-2 -RRB- , mainly in pyramidal neurons . Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter . In all FCD specimens , VEGFA , VEGFB , and both VEGF receptors were highly expressed in dysplastic neurons . IR in astroglial and balloon cells was observed for VEGFA and its receptors . VEGFR-1 displayed strong endothelial staining in FCD . Double-labeling also showed expression of VEGFA , VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage . The neuronal expression of both VEGFA and VEGFB , together with their specific receptors in FCD , suggests autocrine/paracrine effects on dysplastic neurons . These autocrine/paracrine effects could play a role in the development of FCD , preventing the death of abnormal neuronal cells . In addition , the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions . Introduction The vascular endothelial growth factor -LRB- VEGF -RRB- family includes seven members which are structurally homologous , but display molecular and functional diversity -LSB- 58 , 81 -RSB- . VEGFA , the most well known member of the VEGF family , is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth , chronic inflammation , and ischemia -LSB- 10 , 16 , 19 -RSB- . In addition to the unquestioned role in angiogenesis , it has recently been shown that VEGFA has direct trophic effects on neuronal and glial cells in the central nervous system -LSB- 10 , 22 , 25 , 53 -RSB- . VEGFB is most closely related to VEGFA -LSB- 46 , 81 -RSB- ; however , the biological function of VEGFB is less well characterized than the function of VEGFA . VEGFB is expressed early during development and appears to have prominent expression in the central nervous system -LSB- 1 , 36 -RSB- . Additionally , VEGFB has been shown to function as an angiogenic and neuroprotective protein -LSB- 29 , 63 , 68 -RSB- and recent evidence suggests a role for VEGFB in neurogenesis -LSB- 69 , 70 -RSB- . The diverse functions of VEGF proteins can be explained by their differential binding to signaling VEGF receptors -LSB- VEGFRs ; VEGFR-1 -LRB- Flt-1 -RRB- , VEGFR-2 -LRB- Flk1/KDR -RRB- , and VEGFR-3 -LRB- Flt-4 -RRB- -LSB- 50 , 81 -RSB- -RSB- . VEGFA binds to VEGFR-1 and VEGFR-2 , whereas VEGFB binds specifically to VEGFR-1 and not to VEGFR-2 -LSB- 81 -RSB- . The VEGF-signaling pathway , involving both neuronal and glial cells , has been implicated in several neurological disorders , including neurodegeneration , stroke , and cerebral and spinal trauma -LSB- 10 -RSB- . In addition , expression of VEGFA is upregulated in neuronal and glial cells after epileptic seizures in rats -LSB- 13 -RSB- , suggesting a role for VEGFA in seizure disorders . A recent study points to a neuroprotective role for VEGFA following status epilepticus -LSB- 45 -RSB- . The relevance of these findings in animal models to human epileptic disorders is uncertain . Using serial analysis of gene expression -LRB- SAGE -RRB- , we recently identified the VEGFB gene to be upregulated in human tissue from a patient with focal cortical dysplasia -LRB- FCD -RRB- and intractable epilepsy compared to control cortex -LRB- Boer et al. , unpublished observations -RRB- . Upregulation of VEGFA and its receptor has also been recently shown in the hippocampus of cases of human temporal lobe epilepsy -LRB- TLE -RRB- -LSB- 56 -RSB- . However , the distribution of VEGFA , VEGFB , and VEGFRs in epilepsy-associated human malformations of cortical development has not yet been defined . In the present study , we investigated the expression of both VEGFA and VEGFB and their receptors -LRB- VEGFR-1 and VEGFR-2 -RRB- in patients with FCD , which is a developmental disorder known to be a major cause of intractable epilepsy -LSB- 73 -RSB- . We report the specific cellular distribution , including both the neuronal and the glial components of the dysplastic cortex , and we discuss the potential role of VEGFA , VEGFB , and their receptors in the histogenesis and epileptogenesis of this developmental lesion . Materials and methods Subjects The cases included in this study were obtained from the databases of the Departments of Neuropathology of the Academic Medical Center -LRB- University of Amsterdam ; UVA -RRB- in Amsterdam and the University Medical Center in Utrecht -LRB- UMCU -RRB- . We examined surgically resected tissue from nine patients undergoing epilepsy surgery for focal cortical dysplasia . Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes . The tissue was obtained and used in a manner compliant with the Declaration of Helsinki . The classification system proposed by Palmini et al. -LSB- 48 -RSB- was used for grading the degree of FCD and only patients with FCD type IIB located in the temporal lobe were included . The clinical characteristics derived from the patient 's medical records are summarized in Table 1 . The predominant type of seizure pattern was that of complex partial seizures , which were resistant to maximal doses of antiepileptic drugs -LRB- AEDs ; carbamazepine , valproic acid , phenytoin , levetiracetam , oxcarbazepine , and clonazepam -RRB- . Information concerning the exact time of last seizure occurrence prior to surgical resection was not available . However , all the patients included in our series did not have seizure activity in the last 24 h before surgery . The patients underwent presurgical evaluation -LSB- 74 -RSB- . Intraoperative ECoG was performed routinely in all operations for tailoring of surgery and we classified the post-operative seizure outcome according to Engel -LSB- 17 -RSB- . Follow-up period ranged from 1 to 9 years . Table 1Summary of clinical findings of patients with focal cortical dysplasiaPatient/sex/age -LRB- years -RRB- DiagnosisDuration of epilepsy -LRB- years -RRB- Seizure typeEngel class1/M/11FCD IIB11CPSI2/M/31FCD IIB20CPSI3/F/25FCD IIB9CPSI4/F/22FCD IIB21CPS/SGSI5/M / 18FCD IIB14CPSI6/M/17FCD IIB10CPSI7/F/16FCD IIB11CPSI8/M/29FCD IIB21CPSI9/M/28FCD IIB21CPSIFCD focal cortical dysplasia , CPS complex partial seizures , SGS secondary generalized seizures Normal-appearing control cortex/white matter from temporal region was obtained at autopsy from five adult control patients -LRB- male/female : 2/3 ; mean age 42 , range 17 -- 55 -RRB- without history of neurological diseases . All autopsies were performed within 12 h after death -LRB- post mortem delay : 11 , 11.5 , 9 , 8.5 , 6 -RRB- . The cause of death was represented by acute myocardial infarction . In addition , four of the nine FCD cases contained sufficient amount of perilesional zone -LRB- normal-appearing cortex/white matter adjacent to the lesion -RRB- , for comparison with the autopsy specimens . This material represents good disease control tissue , since it is exposed to the same seizure activity , drugs , fixation time , and the age and gender are also the same . Tissue preparation Tissue was fixed in 10 % buffered formalin and embedded in paraffin . Two representative paraffin blocks per case -LRB- containing the complete lesion or the largest part of the lesion resected at surgery -RRB- were sectioned , stained , and assessed . Paraffin-embedded tissue was sectioned at 6 μm , mounted on organosilane-coated slides -LRB- Sigma , St Louis , MO -RRB- and used for histological and immunocytochemical reactions as described below . Frozen tissue from control cortex and FCD tissue , stored at − 80 °C , was used for western blot analysis . Antibody characterization To document the presence of a heterogeneous population of cells , we used the following antibodies : glial fibrillary acidic protein -LRB- GFAP ; polyclonal rabbit , DAKO , Glostrup , Denmark ; 1:4,000 ; monoclonal mouse , DAKO ; 1:50 -RRB- , vimentin -LRB- mouse clone V9 , DAKO ; 1:1,000 -RRB- , MAP2 -LRB- polyclonal rabbit ; Chemicon ; 1:500 -RRB- , neuronal nuclear protein -LRB- NeuN ; mouse clone MAB377 , Chemicon , Temecula , CA , USA ; 1:2,000 -RRB- , non-phosphorylated neurofilament -LRB- SMI311 ; Sternberger monoclonals , Lutherville , MD ; 1:1,000 -RRB- , human leukocyte antigen -LRB- HLA -RRB- - DP , - DQ , - DR -LRB- CR3/43 ; monoclonal mouse , DAKO ; 1:400 -RRB- , CD68 -LRB- mouse clone PG-M1 , DAKO ; 1:200 -RRB- and CD31 -LRB- mouse clone JC70A , DAKO ; 1:100 -RRB- . For the detection of VEGFA , VEGFB , and their receptors , the following antibodies -LRB- Abs -RRB- were used : VEGFA -LRB- G153-694 , monoclonal mouse ; recognizing VEGF 165 and 189 -LSB- 51 -RSB- , Pharmingen , CA , USA ; 1:100 -RRB- , VEGFA -LRB- A-20 , SC-152 , polyclonal rabbit ; raised against the N-terminus of VEGFA , recognizing VEGF 121 , 165 and 189 , Santa Cruz Bio. , CA , USA ; 1:100 -RRB- , VEGFB -LRB- H-70 , SC-13083 , polyclonal rabbit ; raised against amino acids 1 -- 70 of human VEGFB , Santa Cruz Bio. ; 1:20 -RRB- , Flt-1 -LRB- VEGFR-1 ; C-17 , SC-316 , polyclonal rabbit , Santa Cruz Bio. ; 1:100 -RRB- , Flk-1 -LRB- VEGFR-2 ; A-3 , SC-6251 , monoclonal mouse ; Santa Cruz Bio. ; 1:100 -RRB- . To allow comparative analysis , we used on frozen specimens of normal -LRB- n = 3 -RRB- and FCD tissue -LRB- n = 2 -RRB- , two additional antibodies -LRB- which are not suitable for staining paraffin-embedded , formalin-fixed tissue -RRB- : VEGFR-1 -LRB- clone Flt-19 , 1:400 ; developed against the recombinant human extracellular domain of VEGFR-1 -RRB- and VEGFR-2 -LRB- clone KDR-1 , 1:400 ; developed against the recombinant human extracellular domain of VEGFR-2 -RRB- , kindly provided by Dr. H. A. Weich -LRB- National Research Center for Biotechnology , Braunschweig , Germany -RRB- , and previously characterized on human tissues -LSB- 49 , 64 , 80 -RSB- . Similar immunoreactivity patterns were observed on paraffin-embedded and frozen tissue . The specificity of the antibodies used for immunocytochemistry on paraffin-embedded , formalin-fixed tissue -LRB- VEGFA , VEGFB , VEGFR-1 , VEGFR-2 ; Santa Cruz Bio . -RRB- , was further tested by performing western blot analysis of total homogenates of human control cortex . We also include one FCD case of which sufficient frozen material for blot analysis was available -LRB- Fig. 1 -RRB- . VEGFR1 and VEGFR-2 receptor proteins were detectable as a band of approximately 180 and 200 kDa , respectively ; VEGFB was detectable as a band of approximately 40 kDa ; VEGFA labeled a prominent band at approximately 48 kDa and a light band at 21 kDa -LRB- reducing conditions -RRB- , as recently reported in human brain tissue -LRB- control hippocampus and FCD ; -LSB- 56 -RSB- -RRB- . All immunoreactive bands disappeared after preadsorption with the corresponding peptide . Fig. 1Representative immunoblot of VEGFA , VEGFB , VEGFR-1 , and VEGFR-2 in total homogenates from control cortex and FCD tissue . Expression of β-actin -LRB- as reference protein -RRB- is shown in the same protein extracts For immunoblot analysis , human normal cortex -LRB- n = 3 -RRB- and FCD -LRB- n = 1 -RRB- samples were homogenized in lysis buffer containing 10 mM Tris -LRB- pH 8.0 -RRB- , 150 mM NaCl , 10 % glycerol , 1 % NP-40 , Na-orthevanadate -LRB- 10.4 mg/ml -RRB- , 5 mM EDTA -LRB- pH 8.0 -RRB- , 5 mM NaF , and protease inhibitor cocktail -LRB- Boehringer Mannheim , Germany -RRB- . Protein content was determined using the bicinchoninic acid method -LSB- 65 -RSB- . Non-reducing conditions were used to improve the detection of the VEGFA antibody , as previously reported -LSB- 8 -RSB- . For electrophoresis , equal amounts of proteins -LRB- 30 μg/lane -RRB- were separated by sodium dodecylsulfate-polyacrylamide gel electrophoretic -LRB- SDS-PAGE -RRB- analysis in 7.5 -- 12.5 % gels . Separated proteins were transferred to nitrocellulose paper for 1 h and 30 min , using a semi-dry electroblotting system -LRB- BioRad , Transblot SD , Hercules , CA , USA -RRB- . Blots were incubated overnight in TTBS -LRB- 20 mM Tris , 150 mM NaCl , 0.1 % Tween , pH 7.5 -RRB- / 5 % non-fat dry milk , containing the primary antibody -LRB- VEGFA , VEGFR-1 , and VEGFR-2 , 1:1,000 ; VEGFB , 1:200 -RRB- . After several washes in TTBS , the membranes were incubated in TTBS/5 % non-fat dry milk/1 % BSA , containing the goat anti-rabbit coupled to horseradish peroxidase -LRB- 1:2,500 ; Dako , Denmark -RRB- for 1 h . After washes in TTBS , immunoreactivity was visualized using an enhanced chemiluminescence kit -LRB- Amersham , Buckinghamshire , UK -RRB- . Expression of β-actin -LRB- monoclonal mouse , Sigma , St Louis , MO ; 1:50,000 -RRB- was used as reference protein . Because of the limited availability of frozen material from FCD cases , a complete analysis with statistical comparison between control and FCD by immunoblot could not be performed . Immunocytochemistry Sections were deparaffinized , re-hydrated , and incubated for 20 min in 0.3 % H2O2 diluted in methanol to quench the endogenous peroxidase activity . Antigen retrieval was performed by incubation for 10 min at 121 °C in citrate buffer -LRB- 0.01 M , pH 6.0 -RRB- ; sections were washed with phosphate-buffered saline -LRB- PBS -RRB- , and incubated for 30 min in 10 % normal goat serum -LRB- Harlan Sera-Lab , Loughborough , Leicestershire , UK -RRB- . We incubated the sections with the primary antibodies overnight at 4 °C . Hereafter , sections were washed in PBS and we used the ready-for-use Powervision peroxidase system -LRB- Immunologic , Duiven , The Netherlands -RRB- and 3,3 ′ - diaminobenzidine as chromogen to develop the staining . Sections were counterstained with hematoxylin , dehydrated , and coverslipped . Sections incubated without the primary antibody and excess of the antigenic peptide were essentially blank . For double-labeling studies , after incubation overnight at 4 °C with the primary antibodies , sections were incubated for 2 h at RT with Alexa Fluor ® 568-conjugated anti-rabbit IgG and Alexa Fluor ® 488 anti-mouse IgG -LRB- 1:1,000 ; Molecular Probes , The Netherlands -RRB- . The VEGFR-2 antibody -LRB- monoclonal mouse -RRB- could only be combined with MAP2 and GFAP -LRB- polyclonal rabbit -RRB- . Sections were analyzed by means of a laser scanning confocal microscope -LRB- Bio-Rad , Hercules , CA , USA ; MRC1024 -RRB- equipped with an argon-ion laser . Evaluation of immunostaining Semi-quantitative evaluation of immunoreactivity As previously reported -LSB- 3 , 54 -RSB- , a semi-quantitative analysis was done using an Olympus microscope and examining in each section , high-power non overlapping fields -LRB- of 0.0655 × 0.0655 mm width , each corresponding to 4.290 μm2 -RRB- , defined in the center of the lesion using a square grid inserted into the eyepiece . A total microscopical area of 858.050 μm2 was assessed per case . Neuronal cell bodies were differentiated from glia and glia-neuronal balloon cells on the basis of morphology . Balloon cells have eccentric nuclei and ballooned opalescent eosinophilic cytoplasm . The staining intensity was evaluated using a semi-quantitative three-point scale where immunoreactivity was defined as : − absent -LRB- 0 -RRB- ; + moderate -LRB- 1 -RRB- ; + + strong staining -LRB- 2 -RRB- ; intensity score -LRB- Table 2 -RRB- . This score represents the predominant cell staining intensity found in each section for the different cell types -LRB- neurons , astrocytes , microglial cells , and balloon cells -RRB- as averaged from the selected fields -LRB- as previously described -LSB- 3 , 54 -RSB- -RRB- . Table 2VEGFA , VEGFB , VEGFR-1 , and VEGFR-2 distribution in different cellular types in cases of FCD -LRB- % of cases with immunoreactive cells -RRB- Focal cortical dysplasia -LRB- n = 9 -RRB- NeuronsAstrocytesBalloon cells − + + + − + + + − + + + VEGFA022 % 78 % 011 % 89 % 011 % 89 % VEGFB033 % 67 % 78 % 22 % 078 % 22 % 0VEGFR-1055 % 45 % 022 % 78 % 033 % 67 % VEGFR-2011 % 89 % 55 % 45 % 022 % 45 % 33 % FCD focal cortical dysplasia ; immunoreactivity : − not present , + moderate , + + strong Frequency of cell staining In each slice , we assessed the number of neurons and astrocytes labeled by a specific Ab on the total number of each cell type within the lesion using an ocular grid -LSB- 4 -RSB- . This frequency score was assigned using three distinct categories : -LRB- 1 -RRB- < 10 % , rare ; -LRB- 2 -RRB- 11 -- 30 % , sparse ; -LRB- 3 -RRB- > 30 % , high . The product of the intensity and the frequency scores was taken to give the total immunoreactivity score , as previously reported -LSB- 23 , 54 -RSB- . For statistical analysis of data , SPSS for Windows was used . Data were compared using a non-parametric Kruskal -- Wallis test followed by a Mann -- Whitney test to assess the difference between groups . P < 0.05 was taken as the level of significance . Results Human material and histological features All nine patients had chronic pharmacoresistant epilepsy and were all seizure-free postoperatively -LRB- Engel 's class I ; Table 1 -RRB- . The FCD cases included in this study have all the histopathological features of severe -LRB- type IIB -RRB- FCD , according to the classification of Palmini et al. -LSB- 48 -RSB- . The resected specimens consisted of disorganized neocortex containing immature neurons , giant neurons , dysmorphic neurons , and balloon cells . Neurons and balloon cells were also observed in the subcortical white matter and there was a prominent population of reactive astrocytes . Cells of the microglia/macrophage lineage were also observed within the dysplastic cortex , suggesting activation of inflammatory processes in FCD -LSB- 6 -RSB- . Expression of VEGF and VEGFR in normal temporal cortex and FCD Cellular distribution of VEGFA VEGFA staining was observed within the histologically normal cortex -LRB- Fig. 2a , b -RRB- . The staining was strongest in pyramidal neurons , which displayed somatic staining and staining of the apical dendrites -LRB- Fig. 2b -RRB- . Neuropil staining was weak and resting glial cells did not show VEGFA immunoreactivity -LRB- IR -RRB- . Weak staining was observed in endothelial cells . Autopsy material and the perilesional cortex showed similar IR . Fig. 2VEGFA immunoreactivity in focal cortical dysplasia type IIB . a VEGFA immunoreactivity -LRB- IR -RRB- within the histologically normal adult cortex . b High magnification showing somatic staining in pyramidal neurons -LRB- inset : vascular staining -RRB- . c VEGFA in focal cortical dysplasia -LRB- FCD ; type IIB -RRB- showing strong IR within the dysplastic cortex . d High magnification showing VEGFA IR in dysplastic neurons -LRB- arrows -RRB- . e A binucleated VEGFA positive dysplastic neuron . f VEGFA IR within the subcortical dysplastic region . g VEGFA expression in a balloon cell . h , i VEGFA expression in reactive astrocytes -LRB- arrows in i indicate perivascular astrocytic end-feet -RRB- j -- l double-labeling of GFAP -LRB- green , j -RRB- with VEGFA -LRB- red , k -RRB- shows co-localization -LRB- yellow , l -RRB- in astrocytes . m -- o Double-labeling of non-phosphorylated neurofilament -LRB- SMI311 ; NF , green , m -RRB- with VEGFA -LRB- red , n -RRB- shows co-localization -LRB- yellow , o -RRB- in dysplastic neurons . p Merged image showing co-localization of CD68 -LRB- green -RRB- with VEGFA -LRB- red -RRB- in macrophages . Scale bar in a a , c , f 400 μm ; b , d 120 μm ; e , g -- i 35 μm ; j -- o 40 μm ; p 18 μm In the majority of FCD cases , strong VEGFA immunoreactivity -LRB- IR -RRB- was observed in dysplastic neurons located throughout the dysplastic cortex -LRB- Fig. 2c -- e ; Table 2 ; Fig. 6 -RRB- . Strong staining was also detected in balloon cells , reactive astrocytes , and in perivascular astrocytic end-feet -LRB- Fig. 2f -- i ; Table 2 ; Fig. 6 -RRB- . Endothelial IR was weak . Double-labeling experiments confirmed expression in reactive astrocytes , neurons , and in CD68 + macrophages -LRB- Fig. 2p -RRB- . Immunocytochemistry using two different antibodies to VEGF -LRB- Pharmingen and Santa Cruz Bio . -RRB- demonstrated similar patterns . Cellular distribution of VEGFB Histologically normal cortex displayed only weak VEGFB IR -LRB- Fig. 3a -RRB- . Both autopsy and surgical specimens showed light staining in pyramidal neurons and in endothelial cells . Glial cells did not show VEGFB IR . Fig. 3VEGFB immunoreactivity in focal cortical dysplasia type IIB . a Histologically normal adult cortex , showing neuronal distribution of VEGFB with weak immunoreactivity -LRB- IR -RRB- in pyramidal cell neurons -LRB- high magnification of a pyramidal neuron is shown in the inset aI -RRB- ; weak staining was also observed in blood vessels -LRB- inset aII -RRB- . b VEGFB in focal cortical dysplasia -LRB- FCD ; type IIB -RRB- showing strong IR within the dysplastic cortex . c Strong VEGFB IR in dysplastic neurons of different size and shape -LRB- arrows -RRB- . d Undetectable VEGFB IR in balloon cells -LRB- arrows -RRB- . e -- g Absence of co-localization between GFAP -LRB- green , e -RRB- with VEGFB -LRB- red , f -RRB- in astrocytes -LRB- g , merged image -RRB- . h -- j Double-labeling of non-phosphorylated neurofilament -LRB- SMI311 ; NF , green , h -RRB- with VEGFB -LRB- red , i -RRB- shows co-localization -LRB- yellow , j -RRB- in dysplastic neurons . k Merged image showing co-localization of CD68 -LRB- CD68 ; green -RRB- with VEGFB -LRB- red -RRB- in macrophages . Scale bar in a a , b 200 μm ; c -- j 40 μm ; k 18 μm In FCD specimens , moderate to strong VEGFB IR was observed within the dysplastic cortex -LRB- Fig. 3b ; Table 2 ; Fig. 6 -RRB- with strong VEGFB IR in dysplastic neurons -LRB- Fig. 3c -RRB- . In the majority of cases -LRB- seven out of nine -RRB- , balloon cells and reactive astrocytes did not express VEGFB -LRB- Fig. 3d ; Table 2 ; Fig. 6 -RRB- . Double-labeling experiments confirmed the absence of VEGFB IR in GFAP-positive cells -LRB- astrocytes -RRB- , whereas co-localization was found with neurofilament in dysplastic neurons -LRB- Fig. 3h -- j -RRB- . VEGFB IR was also observed in CD68 + macrophages -LRB- Fig. 3k -RRB- . Cellular distribution of VEGFR-1 Histologically normal cortex -LRB- autopsy and surgical specimens -RRB- displayed only weak VEGFR-1 IR , which was restricted to pyramidal neurons -LRB- Fig. 4a -RRB- . IR in blood vessels was weak -LRB- Fig. 4b -RRB- . Glial cells did not show VEGFR-1 IR . Fig. 4VEGFR-1 immunoreactivity in focal cortical dysplasia type IIB . a , b Histologically normal adult cortex -LRB- a -RRB- and white matter -LRB- b -RRB- showing weak immunoreactivity -LRB- IR -RRB- in neurons -LRB- a -RRB- and blood vessels -LRB- b ; arrowheads -RRB- . c , d VEGFR-1 in focal cortical dysplasia -LRB- FCD ; type IIB -RRB- showing strong IR in dysplastic neurons -LRB- c ; arrows -RRB- and in blood vessels -LRB- d ; arrows -RRB- . Inset in c Co-localization between non-phosphorylated neurofilament -LRB- SMI311 ; NF , green -RRB- with VEGFR-1 -LRB- red -RRB- in dysplastic neurons . Inset in d Co-localization between CD31 -LRB- endothelial marker ; green -RRB- with VEGFR-1 -LRB- red -RRB- in blood vessels . e -- g Strong IR in balloon cells of different size -LRB- arrows -RRB- and glial cells -LRB- arrowheads in e -RRB- . h -- j Co-localization between GFAP -LRB- green , h -RRB- with VEGFR-1 -LRB- red , i -RRB- in astrocytes -LRB- j , merged image -RRB- . k -- m Co-localization of CD68 -LRB- CD68 ; green , k -RRB- with VEGFR-1 -LRB- red , l -RRB- in macrophages -LRB- m , merged image -RRB- . Scale bar in a a -- d and h -- m 40 μm ; e -- g 35 μm In FCD specimens , moderate to strong VEGFR-1 staining was observed within the dysplastic cortex -LRB- Fig. 4c -- g ; Table 2 ; Fig. 6 -RRB- . VEGFR-1 IR was observed in different cell types , including dysplastic neurons , astrocytes , and endothelial cells . Double-labeling experiments confirmed the co-localization of VEGFR-1 IR with neuronal -LRB- inset in Fig. 4c -RRB- , endothelial -LRB- inset in Fig. 4d -RRB- , and glial -LRB- Fig. 4h -- j -RRB- markers . Additionally , expression of VEGFR-1 was observed in CD68 + macrophages -LRB- Fig. 4k -- m -RRB- . Cellular distribution of VEGFR-2 VEGFR-2 staining was observed within the histologically normal cortex -LRB- autopsy and perilesional zone -RRB- in pyramidal neurons -LRB- Fig. 5a -RRB- . IR in blood vessels was weak -LRB- Fig. 5b -RRB- . Glial cells did not show VEGFB IR . Fig. 5VEGFR-2 immunoreactivity in focal cortical dysplasia type IIB . a , b Histologically normal adult cortex -LRB- a -RRB- and white matter -LRB- b -RRB- showing moderate immunoreactivity -LRB- IR -RRB- in pyramidal cells -LRB- a -RRB- ; in the white matter , endothelial IR was weak and glial IR was under detection level -LRB- b -RRB- . c , d VEGFR-2 in focal cortical dysplasia -LRB- FCD ; type IIB -RRB- showing strong IR in dysplastic neurons -LRB- arrows in c -RRB- , but weak IR in blood vessels -LRB- arrowheads in d -RRB- . e , f Strong IR in a dysplastic neuron -LRB- arrow in e -RRB- and in a balloon cell -LRB- arrow in f -RRB- , but weak IR in glial cells -LRB- arrowheads in e , f -RRB- . Inset in e Co-localization between non-phosphorylated neurofilament -LRB- SMI311 ; NF , green -RRB- with VEGFR-2 -LRB- red -RRB- in a dysplastic neuron . g -- i Co-localization between MAP-2 -LRB- green , g -RRB- with VEGFR-2 -LRB- red , h -RRB- in balloon cells -LRB- i , merged image -RRB- . Scale bar in a a -- d and g -- i 40 μm ; e , f 35 μm In the large majority of FCD cases -LRB- eight out of nine -RRB- , VEGFR-2 was strongly expressed in dysplastic neurons -LRB- Fig. 5c , e ; Table 2 ; Fig. 6 -RRB- . VEGFR-2 IR was also detected in balloon cells , but only three out of nine cases displayed strong staining for VEGFR-2 -LRB- Fig. 5f ; Table 2 -RRB- . Endothelial expression was weak . In many FCD cases -LRB- five out of nine -RRB- , expression of VEGFR-2 was undetectable in reactive astrocytes -LRB- Table 2 -RRB- . Double-labeling experiments confirmed the co-localization of VEGFR-2 IR with neuronal markers -LRB- neurofilament or MAP2 , Fig. 5e , g -- i -RRB- within the dysplastic cortex . Fig. 6Distribution of immunoreactivity scores -LRB- total score ; see details in `` Materials and methods '' section -RRB- in neurons and astrocytes of control , perilesional , and FCD specimens . a , e VEGFA ; b , f VEGFB ; c , g VEGFR-1 ; d , h VEGFR-2 . a -- d Neurons ; e -- h astrocytes . IR scores of VEGFs and VEGFRs in neurons of FCD were greater than IR scores of VEGFs and VEGFRs of control and perilesional cortex ; scores of VEGFA and VEGFRs in astrocytes of FCD were greater than IR scores of VEGFA and VEGFRs of control and perilesional cortex -LRB- P < 0.05 -RRB- . There were no significant differences in IR scores of VEGFs and VEGFRs between control and perilesional cortex Discussion In addition to their role in angiogenesis , VEGF proteins and their receptors have been implicated in several neurological disorders , including epilepsy -LSB- 13 , 44 , 56 -RSB- . In the present study , we demonstrate a prominent expression of VEGFA , VEGFB , and their signaling receptors in FCD type IIB , a malformation of cortical development associated with intractable epilepsy . This is particularly interesting in view of the recently proposed role of VEGFs and their signaling pathways during development and in epilepsy-associated pathologies -LSB- 13 , 26 , 33 , 69 , 76 -RSB- . Expression of VEGFA and VEGFB in normal temporal cortex In histologically normal temporal cortex -LRB- autopsy and perilesional zone -RRB- , we have shown weak expression of both VEGFA and VEGFB in cortical neurons . Expression of VEGFA and VEGFB , including both mRNA and protein , has been demonstrated in neurons in adult rodent brain -LSB- 39 , 41 , 42 , 72 -RSB- . In human adult brain , only few studies have described neuronal expression of VEGFA in control tissue -LSB- 8 , 78 -RSB- , which was similar to our observed staining pattern in the control temporal specimens . To our knowledge , previous studies of VEGFB protein expression in human control cortex have not been described . However , VEGFB mRNA was detected in human hippocampal cortex -LSB- 24 -RSB- , and VEGFB mRNA and protein expression has been described in adult rodent brain -LSB- 36 , 42 -RSB- . In control rat brain , VEGFB was constitutively expressed in endothelial cells -LSB- 42 -RSB- . In our study , we observed only weak endothelial VEGFB IR in blood vessels . In agreement with previous studies -LSB- 8 , 36 , 78 -RSB- , immunoreactivity -LRB- IR -RRB- for both VEGFs was not observed in glial cells within control specimens . Differential cellular distribution of VEGFA and VEGFB in FCD In the present study , we provide evidence for a consistent expression of both VEGFA and VEGFB within the dysplastic cortex of patients with FCD . Both VEGFs are highly expressed in dysplastic neurons ; however , only the VEGFA protein is prominently expressed in reactive astrocytes . Expression of VEGFA in astrocytes has been shown in several other pathologies associated with reactive gliosis , such as ischemic stroke , traumatic brain injury , neurodegenerative disorders , and the hippocampus following entorhinal deafferentation -LSB- 8 , 27 , 59 , 78 , 79 -RSB- . In addition , we previously reported upregulation of both neuronal and glial VEGFA expression in patients with hemimegalenchephaly , an epilepsy-associated malformation of cortical development -LSB- 5 , 7 -RSB- . In the present study , we also observed expression of VEGFA in balloon cells , which are characteristic cell types of severe FCD -LSB- 48 -RSB- . Whether these cells are glial or neuronal in nature is still controversial -LSB- 12 -RSB- . Induction of both neuronal and astroglial VEGFA expression has been shown in different experimental models of seizures and human temporal lobe epilepsy -LRB- TLE -RRB- -LSB- 13 , 44 , 56 -RSB- . Rigau et al. -LSB- 56 -RSB- showed increased levels of VEGFA in the hippocampus of several cases of TLE , including two cases of focal dysplasia . All TLE cases showed VEGFA expression in pyramidal neurons and granule cells of the hippocampus -LSB- 56 -RSB- . However , immunocytochemical analysis of the temporal cortex and the FCD cases was not performed . The molecular mechanism underlying the induction of VEGFA expression after seizures remains unclear . One possible mechanism , which has been proposed to explain the association between seizure activity and the induction of VEGFA expression , is represented by the stabilization of the hypoxia inducible factor-1α -LRB- HIF-1α -RRB- . HIF-1α is a transcription factor which upregulates VEGFA transcription under hypoxic conditions -LSB- 21 , 38 , 61 -RSB- . Hypoxia may occur during seizures , representing an important trigger in the induction of VEGFA expression , particularly in case of long lasting seizures , such as in status epilepticus models . However , VEGFA expression is already induced after acute seizures -LSB- 44 -RSB- and the mechanisms that regulate VEGFA expression are complex . Several transcription factors , including AP-1 , HIF-1α , and NF-κB , have been identified to regulate VEGFA expression -LSB- 31 -RSB- , and recently it has also been shown that inflammatory cytokines , such as interleukin-1β -LRB- IL-1β -RRB- , activate HIF1α and VEGFA gene expression in primary human astrocytes -LSB- 2 -RSB- . Interestingly , increased expression of proinflammatory cytokines and related molecules has been reported in both animal models and human epilepsy-associated pathologies , including FCD -LSB- 3 , 15 , 54 , 77 -RSB- . In addition , VEGFA has been demonstrated to be a key mediator of the inflammatory process -LSB- 14 , 35 -RSB- . Thus , we might speculate that the prominent expression of VEGFA within the dysplastic cortex could be a critical component of the complex cascade of events leading to a chronic inflammatory state and the sustained seizure activity -LSB- 76 , 77 -RSB- . With respect to inflammation , inflammatory cells , such as macrophages , can also release various angiogenic cytokines including VEGFs -LSB- 71 -RSB- . Accordingly , we observed expression of both VEGFA and VEGFB in macrophages -LRB- CD68-positive cells -RRB- , as previously shown in animal models of brain ischemia -LSB- 11 , 42 , 52 -RSB- . VEGFA effects can also compromise the integrity of the blood -- brain barrier -LRB- BBB ; -LSB- 60 -RSB- -RRB- . Interestingly , alterations of the BBB permeability have been recently observed in both human and experimental temporal lobe epilepsy with positive correlation between the increased vascular permeability and the occurrence of spontaneous seizures in chronic epileptic rats -LSB- 55 , 56 , 75 -RSB- . In contrast , several studies highlight a dichotomous function of VEGFA , also demonstrating a neuroprotective role -LSB- 13 , 47 -RSB- . Administration of VEGFA and neuronal expression of VEGFA have been shown to stimulate neurogenesis in vitro and in vivo -LSB- 9 , 28 -RSB- . In addition , it has been suggested that the neuroprotective effects of VEGFA are mediated by the neuronal VEGFR-2 and the subsequent activation of the PI3K/Akt survival pathway -LSB- 32 , 67 -RSB- . VEGFB expression is not induced by hypoxia or several transcription factors known to regulate VEGFA expression -LSB- 18 -RSB- , as the promoter region of VEGFB lacks HIF-1 and AP-1 sites -LSB- 43 , 62 -RSB- . The regulation of the expression of VEGFB remains unknown . Since all cases examined were associated with epilepsy , we can not exclude that chronic seizure activity could also contribute to the VEGFB expression in FCD specimens . Alternatively , since VEGFB expression has been shown to be prominent during early brain development -LSB- 36 -RSB- , the strong neuronal expression of VEGFB could represent an intrinsic and immature feature of the dysplastic neuronal cells that could contribute to their survival . Recent studies using VEGFB knock-out mice demonstrate a neurotrophic and neuroprotective activity of VEGFB , exerting a direct action on neurons , and promoting neurogenesis -LSB- 68 , 69 -RSB- . This is an observational study and we were , therefore , not able to investigate the spatio-temporal regulation of the VEGF system . Further research in animal models of cortical dysplasia is clearly needed to elucidate the role of VEGFs and their signaling pathways in the histogenesis or epileptogenesis of developmental disorders . Expression of VEGF receptors in normal temporal cortex In histologically normal temporal cortex -LRB- autopsy and perilesional zone -RRB- , VEGFR-1 and VEGFR-2 showed a similar pattern of expression , with weak to moderate immunostaining in pyramidal neurons . Neuronal expression of VEGFRs ' mRNA and protein has been reported in adult human and rodent brain , with strong expression in the hippocampus -LSB- 8 , 11 , 78 , 79 -RSB- . In agreement with these studies , we did not detect glial VEGFR expression in histologically normal cortex and only weak VEGFR expression was observed in endothelial cells . Differential cellular distribution of VEGFR-1 and VEGFR-2 in FCD Consistent expression of both VEGFR-1 and VEGFR-2 was detected within the dysplastic cortex of patients with FCD . Both receptors were upregulated in dysplastic neurons . Increased expression of the VEGFRs and , in particular VEGFR-1 , was observed in reactive astrocytes . Upregulation of VEGFRs in neurons and reactive astrocytes has been shown in several other pathological conditions including ischemia , neurodegenerative diseases , and trauma -LSB- 8 , 11 , 37 , 39 , 66 , 79 -RSB- . Recently , increased expression of VEGFR-2 has been shown in several cases of TLE , including two cases of cortical dysplasia -LSB- 56 -RSB- . Immunocytochemical analysis demonstrated expression only in endothelial cells , whereas neuronal VEGFR-2 IR was not detected in either control hippocampus or TLE specimens -LSB- 56 -RSB- . Differences in the phenotypes of cells expressing VEGFRs have been observed in several other studies -LSB- 8 , 30 , 52 -RSB- . These discrepancies may be caused by differences in experimental methods , tissue processing , or the use of different antibodies that recognize different epitopes . Our results support the neuronal expression of VEGFR-2 recently reported in human brain -LSB- 8 -RSB- . The similar expression pattern of VEGFA and VEGFR-2 , with prominent neuronal IR , suggests autocrine/paracrine effects on dysplastic neurons , supporting the hypothesis of a mechanism to protect abnormal neurons from cell death associated with seizures . Autocrine and/or paracrine effects of VEGFA are supported by the observation that administration of VEGFA has been shown to induce mRNA and protein expression of both receptors in adult rat brain -LSB- 34 , 57 -RSB- . A protective mechanism of VEGFA has been suggested in epileptic rats , showing that VEGFA may reduce spontaneous discharges in epileptic rats -LSB- 40 -RSB- . Therefore , upregulation of VEGFA could represent an endogenous compensatory mechanism to reduce excitability and to prevent cell loss after severe seizures . Accordingly , infusion of VEGFA into the hippocampus has been shown to protect against neuronal cell loss after pilocarpine-induced status epilepticus -LSB- 45 -RSB- . In the present study , we also provide evidence for the expression of VEGFR-1 in activated cells of the microglia/macrophage lineage , which have been shown to be present in FCD specimens -LSB- 6 -RSB- . This is in agreement with previous in vitro and in vivo studies -LSB- 11 , 20 -RSB- showing VEGFR-1 expression in activated microglial cells . These observations suggest that the microglia/macrophage lineage is also a target for VEGF , which may affect chemotaxis and proliferation of these cells , contributing to the inflammatory state in the epileptic brain . There is substantial information about the function and the signaling through VEGFR-2 ; in contrast , signaling through VEGFR-1 remains poorly understood and has been a matter of discussion . A decoy role has been proposed for VEGFR-1 , but more recently , functional signaling via VEGFR-1 has been reported -LRB- for reviews see -LSB- 43 , 67 -RSB- -RRB- . These observations may give rise to new therapeutic strategies focusing on VEGFR-1 specific ligands , such as VEGFB -LSB- 43 -RSB- . Although a rapid induction of VEGF and its receptors has been shown in different experimental models of seizures -LSB- 13 , 44 , 56 -RSB- , seizures alone can not account for changes in neuronal and glial expression in FCD since perilesional tissue was exposed to seizures but did not show significant upregulation of VEGFs and/or VEGFRs . Therefore , the lesion per se or the concomitant presence of the lesion and the epileptic activity , are likely to play a role in modulating the VEGF system in these developmental disorders . Conclusions Our observed cellular distribution of VEGFA , VEGFB , and their signaling receptors indicate that different cellular components of FCD are involved in VEGF-signaling . In this context , future studies , using both in vivo and in vitro models , will be important to achieve a better understanding of the role of the VEGF-mediated pathways in the histogenesis and epileptogenesis of developmental lesions associated with intractable chronic epilepsy . Presently , signaling via VEGF receptors is not targeted by existing therapies in epileptic patients , but it can be potentially useful in view of its involvement in the regulation of neurogenesis , inflammation , and BBB integrity . However , an effective therapeutic intervention based on modulation of the VEGF system has to take into consideration the specific role of VEGFA and VEGFB and the multiple effects -LRB- protective and/or detrimental -RRB- reported for VEGFA .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

vascular endothelial growth factor
receptors
dysplastic neurons
astrocytes
immunocytochemistry
epilepsy