Cholinergic imbalance in the multiple sclerosis hippocampus

Post-mortem brain material was obtained from the Netherlands Brain Bank, Amsterdam, The Netherlands. From 15 MS patients, as well as 10 donors without neurological disease, and 10 AD patients, frozen, coronally cut hippocampal tissue was selected. From a subset of the cases (i.e., 7 controls, 11 MS, and 8 AD), paraffin-embedded material was also available. Non-neurological controls were carefully matched to the MS patient samples for post-mortem delay, age, sex and Braak stage. Upon neuropathological assessment, the AD cases had Braak scores [5] of 5 or higher and were included to serve as a positive control regarding possible cholinergic changes. Clinical and pathological data of the MS patients, non-neurological controls and AD patients are provided in Table 1.

The study was approved by the institutional ethics review board (VU University Medical Center, Amsterdam) and all donors or their next of kin provided written informed consent for brain autopsy and use of material and clinical information for research purposes.

The hippocampal region, including Cornu Ammonis (CA) 1-4, identified on sections stained for proteolipid protein (PLP) (see “Immunohistochemistry”) were traced back in the tissue blocks and carved with a scalpel. In order to ensure that only the correct hippocampal areas were isolated for an adequate comparison, cryosections were cut before and after tissue isolation and subsequently stained for PLP. Tissue for biochemical assays and western blot analyses were isolated separately and were stored at −80°C until subsequent analyses.

As described previously [16], PLP-stained hippocampal tissue sections were scored for the presence of lesions according to their anatomical localization. Lesions were classified as (1) mixed intrahippocampal-perihippocampal lesions; and (2) isolated intrahippocampal lesions (i.e. specified for CA subfield) and subsequently counted. Furthermore, microglial activation, based on anti-human leukocyte antigen-DR (HLA-DR) staining (see “Immunohistochemistry”) was also examined.

Hippocampal tissue homogenates were blinded and samples were analyzed for protein content by the Bradford protein assay. Subsequently, a ChAT enzymatic activity assay was performed following the technique used by Fonnum [14] with minor modifications to the protocol [24]. Frozen hippocampal tissue which was stored at −80°C was homogenised in ice cold homogenization buffer (0.87 mM EDTA, 0.1% Triton X-100, pH 7.0). Samples were analyzed for protein content by Bradford protein assay. An incubation mixture (0.4 mM ¹⁴C-acetyl CoA, 1 mg/ml BSA, 0.3 mM eserine salicylate, 0.6 M NaCl, 4 mM choline chloride, 0.1 M NaH₂PO₄, 17.35 mM EDTA) was prepared on ice. The experiments were set up with two time points. Each sample was prepared in triplicate. In addition, a total radioactivity control was set up consisting of incubation buffer only. A blank control was set up with only extraction buffer present. For the first time point brain homogenate was added to incubation buffer with a 1:1 ratio, using equal volumes (e.g. 10 μl) for each sample and incubated at 30°C. Just prior to the end incubation, brain homogenate samples were added to incubation buffer for without incubation. All reactions were then quenched with the addition of ice cold dH₂O. 1 ml of extraction buffer (15% v/v acetonitrile, 85% toluene, 0.5% w/v sodium tetraphenylboron) was added to the experimental samples. Eppendorfs were spun at 13,000 rpm for 2 min. 650 μl of the upper phase was transferred into scintillant tubes. Scintillant tubes for the controls were prepared using the blank and total samples. 2 ml of liquid scintillant (OptiPhase ‘HiSafe’, Perkin Elmer, UK) was added. Tubes were capped and radioactivity was measured in a beta counter, for 10 min each. The activity of ChAT was measured as the amount of acetylcholine produced per hour, per mg of total protein (nmol/h/mg).

On hippocampal cryosections (8 μm thick) acetylcholinesterase histochemistry was performed using a slightly modified version of the direct coloring Karnovsky–Roots method [21]. In short, slides were fixed for 15 min with 8% formaldehyde in 0.1 M CaCl₂ and pre-incubated for 30 min in 0.18 mM tetra-isopropyl-pyrophosphoramide in phosphate buffered saline (iso-ompa) to block aspecific staining. Subsequently, sections were incubated in reaction medium (consisting of 6.5 ml iso-ompa, 5 mg acetyl-thiocholine-iodine, 0.5 ml 1 M sodium citrate, 1.0 ml 30 mM copper(II)sulphate, 1.0 ml distilled water and 1.0 ml 5 mM potassium ferricyanide) at 37°C for 6 h to visualise acetylcholinesterase activity. For orientation cell nuclei were stained with 1% methylgreen.

Five-micrometer thick frozen sections were collected on Superfrost Plus glass slides (VWR international; Leuven, Belgium), dried overnight at room temperature (RT) and subsequently fixed in acetone for 10 min. Then, sections were rinsed for 3 × 10 min with 0.01 M phosphate buffered saline (PBS; pH 7.4) followed by incubation with mouse-anti-human primary antibodies against myelin proteolipid protein (PLP; Clone: Plpc1; mouse IgG2a; 1:500; Serotec, Oxford, UK) diluted in PBS containing 1% bovine serum albumin (BSA) (Roche Diagnostics; Mannheim, Germany) for 1 h. Subsequently, sections were rinsed with PBS (3 × 10 min) and incubated with biotin-labeled rabbit anti-mouse immunoglobulins F(ab′)₂ (1:500) (DAKO, Glostrup, Denmark) diluted in PBS with 1% BSA for 30 min. Then, sections were rinsed in PBS (3 × 10 min) and incubated for 1 h at RT with streptavidin–biotin-peroxidase complexes (1:100; Vectastain; Vector Laboratories Inc., Burlingame, CA, USA). Finally, sections were rinsed with PBS (3 × 10 min) and peroxidase reaction was developed with 3,3′ diaminobenzidine-tetrahydrochloridedihydrate (DAB; DAKO, Glostrup, Denmark) as a chromogen. After a short rinse with tap water, sections were counterstained with hematoxylin for 1 min and intensely washed with tap water for 5 min.

Eight-micrometer thick paraffin sections were deparaffinized in a series of xylene (4 × 5 min), 100% ethanol, 96% ethanol, 70% ethanol. Endogenous peroxidase activity was blocked by incubating the sections in methanol with 0.3% H₂O₂ for 30 min. In case of primary immunostaining with anti-proteolipid protein (PLP) (mouse IgG2a; 1:3,000; Serotec, Oxford, UK) sections were rinsed for 3 × 10 min with PBS followed by incubation with primary antibodies diluted in PBS containing 1% BSA for 1 h. In case of primary immunostaining with anti-HLA-DR, sections were pretreated with microwave antigen retrieval (3 min at 900 W and 30 min at 180 W). After pretreatment, sections were cooled to room temperature, rinsed with PBS (3 × 10 min) and incubated with anti-HLA-DR (mouse IgG2b; 1:50, generous gift from Dr. Hilgers, VUMC, Amsterdam) diluted in PBS containing 1% BSA for 1 h. Then, sections were rinsed again with PBS (3 × 10 min) and incubated with EnVision horseradish peroxidase (HRP) complex (DAKO, Glostrup, Denmark) and finally with 3,3′ diaminobenzidine-tetrahydrochloridedihydrate (DAB; DAKO, Glostrup, Denmark) as a chromogen. After a short rinse with tap water, sections were counterstained with hematoxylin for 1 min and intensely washed with tap water for 5 min.

For immunostaining with anti-ChAT (AB144P, goat polyclonal, 1:400, Millipore, Billerica, MA, USA) sections were deparaffinized and rehydrated as described above. Subsequently, sections were incubated with 37% formaldehyde for 10 min and then rinsed with distilled water (2 × 5 min). Then, sections were rinsed with citrate buffer (pH 6.0) (3 × 10 min) and finally rinsed overnight in citrate buffer at 4°C. The other day the slides were again rinsed for 2 × 10 min with citrate buffer and then 10 min in tris-buffered saline (TBS; pH 7.6). Hereafter, endogenous peroxidase activity was blocked by incubating the sections with TBS with 3% H₂O₂ for 30 min. Then, sections were rinsed with TBS (10 min) and citrate buffer (2 × 10 min). Subsequently, sections were pretreated by microwave antigen retrieval (3 min at 900 W and 30 min at 180 W) in citrate buffer. After pretreatment, sections were cooled to room temperature and rinsed with TBS (3 × 10 min). Then, sections were incubated with 5% normal rabbit serum (DAKO, Glostrup, Denmark) in TBS with 0.5% Triton X-100 (TBS-Tx) for 30 min and then with primary antibodies diluted in TBS-Tx containing 2% normal rabbit serum for 2 h at RT and subsequently 48 h at 4°C. After 2 days, sections were rinsed again with TBS (3 × 10 min) followed by 1 h incubation with biotin-labeled rabbit-anti-goat antibodies (DAKO, Glostrup, Denmark) diluted in TBS-Tx. Subsequently, sections were rinsed in TBS (3 × 10 min) and incubated for 1 h at RT with streptavidin–biotin-peroxidase complexes (1:100; Vectastain; Vector Laboratories Inc., Burlingame, CA, USA) diluted in TBS-Tx. After two rinse steps (10 min) in TBS, sections were rinsed with Tris-hydrochloride (pH 7.6) for 10 min and then incubated with 3,3′ diaminobenzidine-tetrahydrochloridedihydrate (DAB; DAKO, Glostrup, Denmark) as a chromogen. After a short rinse in TBS-hydrochloride (3×), sections were counterstained with hematoxylin for 1 min and intensely washed with tap water for 5 min. For all immunostainings performed, negative controls were prepared by incubation without primary or secondary antibody and gave no immunoreactivity.

For protein analysis, 30 hippocampal sections of 10-μm thick were isolated from 7 controls, 9 MS and 7 AD patients as described in “Sampling procedure”. Subsequently, tissue was homogenized by incubating the samples with radio-immunoprecipation assay (RIPA) buffer and protease inhibitors (Sigma-Aldrich, Zwijndrecht, The Netherlands) on ice for 30 min followed by sonification (Branson sonifier, Danbury, CT, USA). The protein concentration was measured with the bicinchoninic acid (BCA) protein assay (Pierce, Perbio Science, Etten-Leur, The Netherlands) as indicated in the user manual, using bovine serum albumin for standard curve. Samples were stored at −80°C until assayed.

Equal amounts of protein (10 μg) were separated on 10% SDS-PAGE gels (Bio-Rad Laboratories, CA, USA) and transferred to PVDF membranes (Bio-Rad). After blocking in PBS and Odyssey blocking buffer (1:1) (LI-COR Biosciences, Lincoln NE, USA), membranes were incubated with primary antibody anti-AChE (PAB6747, goat polyclonal, 1:2,000, Abnova, Walnut, CA, USA) overnight at 4°C. Then, membranes were rinsed in PBS containing 0.1% Tween-20 (4 × 5 min) and incubated with donkey-anti-goat-IRDye^® 800CW infrared dye (1:15,000) (LI-COR Biosciences) diluted in PBS, Odyssey blocking buffer (1:1), 0.1% Tween-20 and 0.01% SDS for 1 h at RT. Finally, the membranes were rinsed in PBS and 0.1% Tween-20 (4 × 5 min), 3 times in PBS and then membranes were analyzed using a Odyssey Infrared Imaging system (LI-COR Biosciences) and relevant signal intensity was quantified with LI-COR analysis software. Molecular weight and predicted band size of AChE is 68 kDa. After this, the membranes were incubated (overnight at 4°C) with the primary antibody anti-lactate dehydrogenase (1:20,000) (LDH; ab52448, rabbit monoclonal, Abcam, Cambridge, UK). Molecular weight and predicted band size of LDH is 37 kDa. After this, membranes were incubated with goat-anti-rabbit-IRDye^® 800CW infrared dye (1:15,000) (LI-COR Biosciences) diluted in PBS, Odyssey blocking buffer (1:1), 0.1% Tween-20 and 0.01% SDS for 1 h at RT and analyzed as describe above and subsequently relative AChE protein expression was quantified.

(Immuno)histochemically stained sections were scanned with a Mirax slide scanner system (3DHISTECH, Budapest, Hungary). Digital images scanned with a 20× objective were captured in three randomly selected areas of each of the hippocampal subfields (CA1, CA3-2 and CA4) for ChAT immunostainings and AChE histochemical stainings. As already indicated above, paraffin-embedded material was available from 7 control, 8 AD and 11 MS hippocampi and this material was therefore immunohistochemically stained for ChAT. For each investigated hippocampal subfield (i.e. CA1, CA2-3 and CA4), three randomly selected areas were captured for subsequent quantification. This means that for controls a total of 21 (3 × 7), for AD 24 (3 × 8) and for MS 33 (3 × 11) randomly chosen areas were captured. For the control, AD and MS hippocampal sections (frozen) on which AChE enzyme histochemistry was performed, respectively 30 (3 × 10), 30 (3 × 10) and 45 (3 × 15) randomly selected areas from each hippocampal subfield were captured for subsequent quantification. The (immuno)reactivity of the stainings was scored in a blinded fashion with respect to the presence of demyelinated lesions in order to avoid potential bias (e.g. by the expectation that lesional areas should show more pronounced cholinergic system changes). However, differences in terms of cholinergic alterations between lesional and perilesional or non-lesional areas was retrospectively assessed. The relative amount of staining was analyzed using a, for this staining, tailor-made batch mode ImageJ macro as previously described [18]. ImageJ is freely available at: http://​rsb.​info.​nih.​gov/​ij/​index.​html (US National Institutes of Health, Bethesda, MD, USA).