CSF collection
CSF samples were collected after obtaining informed consent as per the institutional human ethics committee guidelines. CSF samples of ALS patients diagnosed using El Escorial criteria [19], were obtained through lumbar puncture. ALS Functional Rating Scale (ALS-FRS) was done to determine the severity of the disease at the time of sample collection. CSF from age and gender matched patients with non-neurodegenerative, non-infectious neurological diseases such as benign intracranial hypertension and peripheral neuropathy were also collected and used as non ALS (NALS-CSF) samples, while CSF samples of patients undergoing orthopaedic surgery without any neurological involvement were used as Normal CSF (N-CSF). Intrathecal procedure was carried out by neurologists and orthopaedic surgeons under aseptic condition. CSF samples were snap frozen in liquid nitrogen and stored at –80°C until further use. Table 1 provides details of CSF samples used for the study.
Enzyme linked immunosorbent assay (ELISA) and enzyme activity of CHIT-1
ELISA for CSF (48 N-CSF, 12 NALS and 158 ALS-CSF) was performed using commercially available kit for CHIT-1 (MBL International, USA) according to manufacturer’s protocol. The CHIT-1 enzyme activity was measured in 45 N-CSF, 10 NALS-CSF and 56 ALS-CSF samples as described previously [5]. In brief, 2.5 μg of total protein was added to 150 μl of 22 μmol 4-methylumbelliferyl β-D-N,N′,N′-triacetylchitotrioside hydrate (Sigma-Aldrich, USA), prepared in 0.5 M citrate-phosphate buffer (pH 5.2). Following incubation for 15 minutes at 37ºC, the reaction was stopped using 100 μl of 0.5 mol Na2CO3-NaHCO3 buffer (pH 10.7). The fluorescence was captured at 365 nm excitation and 450 nm emission using Tecan 2500 fluorimeter (Tecan, USA) and the data was expressed as micromoles of substrate hydrolyzed/μl/min.
CHIT-1 dosage for in vivo studies
The dosage for intrathecal injection of CHIT-1 was based upon the average amount of CHIT-1 present in 5µl of CSF (approximately 90 pg of CHIT-1 as determined by ELISA). The following doses of CHIT-1 were used: 50pg, 100pg, 200pg and 500pg.
In vivo studies
Neonatal Wistar rat pups were obtained from Central Animal Research Facility (CARF), NIMHANS, Bangalore, after obtaining clearance from Institutional Animal Ethics Committee (IAEC). Rat neonates along with lactating mothers were housed at an ambient temperature of 26 ± 2 °C and subjected to the routine light/dark cycle. Lactating mothers had ad libitum access to food and water. Post-natal day3 pups were intrathecally injected with 5 µl of buffer, different doses of recombinant human CHIT-1, N-CSF and ALS-CSF [20-22]. Briefly, rat pups were anesthetized with halothane and a dorsal midline incision (1mm) was made about 1 cm rostral to the base of the tail. Samples were injected into the subarachnoid space with the aid of a micro-injector at a flow rate of 400 nl/min. The needle was retained in its place for 1-2 minutes following injection to prevent back flow of injected sample. The incision was cleaned, sutured and an anti- inflammatory agent, Healex was sprayed on to the sutures. Pups were allowed to recover from anaesthesia and housed with the mother. Pups of both genders were randomly assigned to each of the experimental groups.
Immunohistochemistry
After 48 hours of intrathecal injection, animals were anaesthetized with halothane and perfused transcardially using 4% paraformaldehyde [22, 23]. Spinal cords were dissected out, post fixed in the same fixative for 24 hours and meninges removed. Lumbar region cryoprotected with 30% sucrose was sectioned at 40µ thickness using a cryostat (Leica, Germany).
For immunostaining, antigen unmasking was performed by incubating in sodium citrate buffer (10 mM Sodium citrate, 0.05% tween 20, pH 6.0) for 5 - 10 min at 95 °C and blocked using 3% Bovine Serum Albumin (BSA, Sigma) for 3 hours. Sections were rinsed in 0.1 M PBST and then incubated in the first primary antibody. The sections were again washed and incubated with appropriate fluorescent conjugated secondary antibody. Blocking was done with 3% BSA for 1hour and rinsed and treated subsequently with second primary antibody followed by incubation in second secondary antibody. For Choline Acetyl Transferase (ChAT) staining, spinal cord sections were pretreated with ice cold methanol for 10 min. Lists of antibodies used are given in Table 2.
The fluorescence images of the immunolabelled specimens were analyzed using a Laser confocal scanning microscope (Leica TCS-SL, Germany) with excitation at 488 and 514 nm for FITC and Cy3 respectively. Emission band width of FITC was maintained at 490 – 540nm and 550 – 620nm for Cy3 to prevent overlap of emission frequencies.
Analysis was performed on 10 sections per spinal cord and 3 animals per group were used for the study. Number of ChAT positive motor neurons and Iba1 positive microglia in the ventral horn was counted and the individual means were derived. Area and intensity of GFAP and SMI-31 in white matter was measured and quantified on a scale of 0 (minimum) – 255 (maximum), using an in-built software.
Nissl Staining
Serial sections of the lumbar region of spinal cords post fixed in paraformaldehyde (40 µm) were taken on a vibratome (Leica, Germany), and every fourth section was stained with cresyl violet. Briefly, slides were dipped in chloroform for 1 min, passed in succession through 100% ethanol, 90% ethanol, 80% ethanol and 70%ethanil for 2 min, rehydrated in distilled water for 5 min and then in cresyl violet for 2 min. Slides were allowed to differentiate in diatilled water for 5 min, passed in succession through 70% ethanol for 20 s(× 2), 90% ethanol for 20 s (× 2), 100% ethanol for 1 min (× 2), and xylene for 1 min. The stained slides were dried and mounted with DPX. Images were captured at 10 X with a Leica BX 51 microscope. The number of motor neurons was quantified by using the particle analysis feature of ImageJ. Briefly, the images were converted to 8-bit binary with Huang thresholding, following noise removal and using a size-based filter above 250 μm2 for the analysis [24]. Data was obtained by analysing 5 sections per spinal cord and 3 animals per group were used for the study.
ELISA for pro-inflammatory molecules in tissue lysates
Lumbar region of spinal cord was dissected out 48 hours post intrathecal injection. Tissues were snap frozen and stored at -80oC till use. Samples containing 2% tissue lysate with 1% protease inhibitor were sonicated thrice at 15 Hz for 10 sec. The lysates were centrifuged for 10 min at 5,000 x g and the supernatant was used for ELISA. Samples were protein normalised and 450µg/ml of each sample was used. ELISA for IL-10 and TNFα was performed using commercially available kits (RayBiotech, USA) according to manufacturer’s protocol.
Statistical analysis
Data was expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 and SPSS software. The data was analysed for significance using one-way ANOVA followed by Tukey’s post-hoc test. Receiver operator characteristic (ROC) analysis was performed to assess optimal cut off value and sensitivity and specificity of ELISA. Medcals diagnostic test evaluation calculator was used to calculate predictive values. Pearson correlation was used to find possible correlation between CHIT-1 activity and disease severity on one hand and CHIT-1 level on the other hand. Spearman's correlation was performed to assess correlation between CHIT-1 level and disease duration. It was also performed to correlate enzyme activity of CHIT-1 on one hand and disease severity as well as duration on the other hand. Partial correlation was used to find the relationship between CHIT-I level and disease severity and disease duration.