Background
Hydrocephalus is a common neurological condition where cerebrospinal fluid (CSF) flow dynamics are altered, leading to enlargement of ventricular cavities in the brain. The histopathologic consequences of hydrocephalus depend on the age of onset, rate of ventricular enlargement, and the etiology [
1]. The brain damage induced by hydrocephalus is multifactorial with mechanical factors leading to primary destruction of periventricular axons due to gradual physical stretching and compression, accumulation of metabolic waste products in the CSF, and ischemic changes causing decreased white matter blood flow that contributes to axonal and oligodendroglial damage [
2,
3]. Elevated calcium (Ca
2+) coincides with increased white matter content of calpain I, along with heightened immunoreactivity in periventricular axons exhibited by young hydrocephalic rats with appreciable axonal damage, which suggest that calcium-mediated proteolysis may be associated with axonal cytoskeletal damage found in hydrocephalus [
4].
Magnesium (Mg
2+) is a calcium channel antagonist; extracellular Mg
2+ antagonizes Ca
2+ influx by blocking voltage and receptor-mediated calcium channels along with NMDA channel receptors in a voltage-dependent manner [
5,
6]. Intracellular Mg
2+ is regulated by at least five distinct transporters [
7]. Peripherally administered Mg
2+ enters brain tissue of rabbits and cats, albeit to a lesser magnitude than muscle [
8]. Magnesium sulfate (MgSO
4) administration by bolus or short-term infusion (e.g. 3 days) has shown short-term (hours to <7 days) outcome improvements in animal models of experimental spinal cord injury [
9], reversible focal cerebral ischemia [
10], kainate induced neuron degeneration [
11], hypoxic-ischemic brain damage [
12,
13], and traumatic brain injury [
14‐
16]. Few experiments have looked at longer-term benefits of magnesium. A bolus of magnesium chloride after fluid percussion brain injury in adult rats was associated with improved motor outcome at 4 weeks [
17].
Clinically, MgSO
4 has been used to suppress uterine contractions during premature labor and to reduce convulsions in pregnant women with preeclampsia [
18]. In these circumstances, there is an association between maternal receipt of MgSO
4 and reduced risk of cerebral palsy in the offspring; this suggests that it could be neuroprotective in preterm birth although the benefit in term birth is much less clear [
19,
20]. In infants with hypoxic-ischemic encephalopathy, the composite results of five clinical trials indicate a significant reduction in the unfavorable short-term outcome but a trend to increased mortality overall [
21]. If effective, its protective mechanism remains unclear. MgSO
4 is a vasodilator that might improve cerebral circulation [
22]. It can also reduce monocyte-mediated proinflammatory cytokine production and increase intracellular magnesium levels, which may be beneficial in decreasing inflammation [
23].
Pharmacologic intervention might be a useful supplement to surgical shunts for the management of hydrocephalus [
24]. We previously showed that MgSO
4 was beneficial in hydrocephalic rats [
25]. Three-week old rats received kaolin injections into the cisterna magna to induce hydrocephalus; after 2 weeks, they were treated with parenteral administration of MgSO
4 for 2 more weeks. There was reduced reactive astrogliosis in the frontal cerebrum and improved gait performance on an accelerating rotating cylinder task. Despite these benefits, research with rodents is limiting because they have a small volume of white matter, which is the main region of damage in human brains. Moreover, stroke research has provided a valuable lesson in the major setbacks that can occur when basing clinical treatment on the success of pharmacological studies with rodents; this has prompted the necessity of preclinical treatment efficacy in gyrencephalic brains before proceeding to human trials [
26]. Thus, we developed a model of hydrocephalus in 2-week old ferrets using injection of kaolin (aluminum silicate) into the cisterna magna, and we showed similarities to other animal models and the human condition [
27]. Ferrets are born with relatively immature brains which develop a complex gyrencephalic morphology that has been studied extensively [
28,
29]; consequently they are useful for modeling the neurologic disorders of human fetuses and infants [
30]. Our overall goal is to develop a pharmacologic intervention that could be used to mitigate brain damage in the period prior to definitive shunt therapy. We hypothesized that MgSO
4 treatment would lead to behavioral, structural, and/or biochemical improvements in juvenile ferrets with experimental kaolin-induced hydrocephalus.
Methods
Animals
Twenty pigmented sable ferret kits (n = 13 males and n = 7 females) were obtained from Marshall Farms (North Rose, NY) at postnatal day 7 (P7) in 4 l along with their mothers (jills). The kits stayed with their mothers in enclosed cages until P46. The cages were located in a room kept on a 12:12 h (6 a.m.–6 p.m.) light–dark cycle, and the room temperature and relative humidity were 21–22 °C and ~35–45 %, respectively. Food and water were provided ad libitum; the kits started eating solid food around P30. For identification, tattoos were imprinted on their paws. All animals were treated humanely according to the guidelines set forth by the Canadian Council on Animal Care. The institutional animal ethics committee approved the experimental protocols (protocol #11-012). All efforts were made to minimize the number of animals used and ensure the least amount of suffering experienced.
Hydrocephalus induction
Hydrocephalus was induced using kaolin (aluminum silicate; Sigma, St. Louis MO) as described previously [
27]. Briefly, kaolin injections were performed on all 4 litters at P15 (5 per litter—total n = 20; weight 69–93 g). They were anesthetized using 2.5 % isoflurane in oxygen, and the dorsum of their necks were shaved and cleansed. Using a 27-gauge needle, 0.2 mL of 20 % sterile kaolin suspension (250 mg/mL in 0.9 % saline) was injected percutaneously into the cisterna magna under aseptic conditions. Animals were monitored during recovery and observed for signs of discomfort and/or neurological impairment and then were otherwise returned to their mothers. Subcutaneous (sc) injections of buprenorphine (0.03 mg/kg) and sterile 0.45 % saline were given every 12 h for 2 days to decrease potential pain and possible dehydration, respectively. They were weighed daily, and those experiencing severe neurologic impairment or weight loss were sacrificed to cease further suffering.
Magnetic resonance imaging and assignment to treatment groups
The T2-weighted MR images of the brain were obtained using with a 7 Tesla Bruker Biospec/3 MR scanner (Karlsruhe, Germany) as previously described [
27]. The first images were attained 2 days post kaolin injections at P17 to confirm successful hydrocephalus induction with kaolin, and then 14 days post kaolin at P29 to examine ventricle size and stratify treatment groups. The areas of the lateral ventricles to cerebrum brain area, third ventricle width to cerebrum width, cerebral aqueduct area to midbrain area, and fourth ventricle area to hindbrain area ratios were measured as previously described [
27]. Ferrets were stratified based on ventricle size and assigned listwise to saline (0.9 % NaCl) and MgSO
4 treatment groups. A third set of MR images was taken after the 14-day drug treatment period at P45, no more than 24 h before euthanasia, to assess ventricle size again.
Drug preparation and administration
The MgSO
4 and/or NaCl treatments were administered starting 16 days post-kaolin at P31 (n = 8 for MgSO
4 and n = 8 for NaCl). All animals were weighed daily, and their weights were used to calculate treatment volumes. Stock solutions of 1.0 M MgSO
4 and 0.9 M NaCl were prepared in distilled H
2O at room temperature and stored at 4 °C; the solutions were labeled A and B, and treatments were given blindly based upon a volume drug to body weight formula. They received either a ~9 mM/kg/day dosage of MgSO
4 or NaCl daily for 14 days, which were administered by sc injections given 3 times per day on weekdays (~3 mM/kg/dose) and 2 times per day on weekends (~4.5 mM/kg/dose) to ensure that all animals received the entire dosage. This calculated dosage was derived from testing different MgSO
4 concentrations with rodents in a previous study, where 8.2 mM/kg/day was found to be protective [
25]. The ferrets received their first injection in the morning before behavioral testing. Their second daily injection was given in the late afternoon, and the third injection was administered near midnight to maintain trough levels. Because of the expense, we could not conduct a dose-escalation study.
Behavioral testing
Previous work with kaolin-induced hydrocephalic ferrets [
27] showed that they differed from controls in a limited number of the behavioral tests previously studied [
31]; these were chosen for the current study. Behavioral testing commenced at P10-11 and continued 3 times weekly until P43-44 for all kits (n = 16). Behavior tests were conducted prior to the daily drug administration to minimize the effect of lethargy in the MgSO
4 recipients. The kits were not exposed to the test situations prior to testing. Open field behavior was assessed using two apparatuses. The first chamber was an enclosed square (44 × 43 × 29 cm) with 15 light beam sensors on each axis to quantitate ambulatory, vertical, and total movements (Opto-Varimex 3; Columbus Instruments International Corp., Columbus, OH, USA). Animals were observed individually for 3 min and tested once per session. The second chamber was a 75 × 75 × 45 cm transparent plastic square, where the kits were videotaped, and motor performance was analyzed for 3 min using HVS Image 2100 Plus Tracking System software (HVS Image Ltd, Twickenham, Middlesex, UK). Quantitative measures were performed after dividing the chamber into 100 (7.5 × 7.5 cm) squares and included the length of path traversed and the number and percent of squares entered. Qualitative observations were also recorded in the second chamber for pivoting, crawling, walking, running, and rearing.
Sacrifice and brain dissection
Ferrets were euthanized within 24 h of final MRI using isoflurane anesthesia (5 %) followed by carbon dioxide (CO
2) narcosis and exsanguination by transcardiac perfusion at ~P46 or when humane endpoints were met as described previously [
27]. Ferret brains were rapidly removed, photographed, and then split in the parasagittal plane 1 mm from the midline. The right hemisphere was dissected into anterior frontal lobe, dorsal frontal cerebrum, and dorsal parietal cerebrum, which were frozen in liquid nitrogen (N
2) and stored at −70 °C. The left hemisphere was immersion fixed in cold 3 % buffered paraformaldehyde for several days; then it was sliced in the coronal plane, dehydrated, and embedded in paraffin wax.
Histology and immunohistochemistry
All paraffin blocks were sectioned coronally (6 μm thickness) and stained with hematoxylin and eosin (H and E). The cerebrum at the level of the anterior horn of the lateral ventricles was stained with solochrome cyanine and counterstained with eosin for visualization of myelin. Sections were immunostained with rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:10,000 dilution; DAKO Z0334; Glostrup, Denmark) to label astrocytes and reactive astrocytes. The primary antibody underwent 1.5 h incubation at room temperature. This was followed by incubation with appropriate biotinylated secondary antibody, followed by reaction with streptavidin-peroxidase, detection with diaminobenzidine (DAB, Sigma D5905), and finally counterstaining with hematoxylin. Negative controls were treated without the primary antibody. Corpus callosum thickness was measured using 100× ocular magnification at the sagittal midline and above the lateral angle of the anterior horn of the lateral ventricle. The second site was chosen because fragmentation of the midline region in some hydrocephalic brains preventing proper measurement of corpus callosum thickness. For comparative purposes, non-hydrocephalic ferrets from our previous experiment [
27] were also examined.
Myelin enzymes and enzyme-linked immunosorbent assays
Frozen dorsal frontal and parietal cerebrum samples were homogenized using a radio immunoprecipitation assay (RIPA) buffer including protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and aprotinin, which likely explain the lower myelin enzyme activities than were obtained previously [
27]. Total protein quantification was determined with the Micro BCA (Pierce) Protein Assay kit (Thermo Scientific, Rockford, Illinois, USA). Dorsal frontal cerebrum homogenates were used to quantify myelin basic protein (MBP) and GFAP content by using ELISAs, as previously described in detail [
27]. Colorimetric assays were performed in triplicate, and results are shown in μg of MBP or GFAP per gram of protein and averaged per sample. Dorsal parietal cerebrum samples were used to quantify the enzyme activity of UDP-galactose:ceramide galactosyltransferase (CGalT) and glycerylphosphorylcholine phosphocholine phosphodiesterase (GPC-PP) using the artificial substrate p-nitrophenylphosphorylcholine (pNPP). Both are enzymes that are enriched in myelin. They were quantified in triplicate as described previously [
32,
33].
Atomic absorption spectroscopy
Flame atomic absorption spectroscopy (FAAS) was performed to determine Mg levels in anterior forebrain samples as indicated previously [
34] except for the following differences. Ferrets were given their last dose of MgSO
4 or NaCl 35–152 min (mean 68.88 ± 13.46) before sacrifice. Forebrain samples were thawed and weighed then dissolved in 18 M sulfuric acid and 16 M nitric acid over gentle heat. Standards were prepared from a 1000 μg mL
−1 Mg standard solution (Alfa Aesar 88077) at concentrations of 0.50, 0.40, and 0.20 μg mL
−1, along with a 1 % nitric acid blank. Mg content was measured using Atomic Absorption Spectrometer AAnalyst 400 with a PerkinElmer Lamp (Intensitron) using a wavelength of 285.21 nm. Measurements were performed in triplicate and averaged to obtain concentrations (μg Mg
2+ per gram brain tissue).
Statistical analysis
All data are presented as mean ± SEM, unless otherwise indicated. Quantitative data were analyzed to confirm a normal distribution, and p values ≤0.05 were considered statistically significant. Statistical analyses for the behavioral tasks, MRI, and all biochemical analyses were conducted with the juvenile ferrets (n = 15). Data were assessed using ANOVA and two-tailed t-tests for behavioral testing, ventricle size, histological data, and biochemical assays to compare MgSO4 and NaCl hydrocephalic treatment groups. Qualitative assessments for motor and behavioral development were analyzed separately from quantitative measures. Statistical analyses were conducted using the SPSS 19.0 software program.
Discussion
Mg
2+ was shown to have mild protective benefits in rats with experimental hydrocephalus treated from 5 to 7 weeks age but not in rats treated from 1 to 3 weeks age [
25,
34]. We had hoped that MgSO
4 treatment would also yield therapeutic benefits in young hydrocephalic ferrets. As has been recommended for preclinical stroke and brain trauma studies [
26,
37], the experimental design included randomization, blinding, and multiple outcome measures. However, in comparison to NaCl-treated hydrocephalic ferrets, we did not find any behavioral, histological, or biochemical evidence to support the hypothesis that MgSO
4 therapy, at the same dose that was effective in rats, benefits hydrocephalic ferrets. Treated ferrets had transient sedation, which is well-documented [
38], impaired weight gain, and tendency to greater progression of ventriculomegaly. Despite more severely enlarged ventricles, MgSO
4 treatment was associated with reduced GFAP accumulation in ferrets albeit not significantly; this finding is similar to that seen in hydrocephalic rats treated from 5 to 7 weeks age [
25]. Reduced astroglial reaction has also been reported in kaolin-induced and congenitally hydrocephalic H-Tx rats treated with minocycline or decorin [
39‐
41]. Although reduced GFAP accumulation is often considered an indicator of benefit, another possibility is that Mg
2+, which blocks signaling between astrocytes [
42], simply masks the astrocytic response to brain damage.
Why was MgSO
4 therapy unsuccessful in hydrocephalic ferrets? Rationale for the experiment was based upon a previously-demonstrated neuroprotective effect in juvenile hydrocephalic rats and a range of experimental data from other neurological disorders. Technical and design aspects must be considered. It remains unclear whether entry of Mg
2+ into brain is via the choroid plexus and CSF or through the blood brain barrier [
43,
44], although the observed side effect of sedation indicates entry into the brain [
8,
45,
46]. Lethargy is a potential confounder in the behavioral assessments, and it prevented complete blinding of the investigators. More importantly, weight gain was retarded in the MgSO
4 treated ferrets, possibly because lethargy impaired feeding or because of the effect of Mg
2+ on intestinal smooth muscle [
7]. Undernutrition might have had a negative effect on the outcome. This might be overcome experimentally by using a matched feeding strategy. Unfortunately, ferrets are obligate carnivores with short intestinal tracts; they can require special diets when they are ill [
47], and therefore might not be the ideal animal for studies where feeding is compromised. Periodic subcutaneous injections would result in troughs and peaks; in rats Mg
2+ levels peak at approximately 2 h and return to normal levels within 4 h after injection [
25]. We had considered using osmotic minipumps, but none would accommodate sufficiently large volumes for the treatment period nor could they adjust for the increasing weight of the maturing ferrets. Furthermore, based on discussions with the veterinarians, the potential complications (maternal biting of the surgical site and sloughing of the skin over the minipumps) outweighed the potential benefits. During the treatment period, we did observe that the MgSO
4-treated ferrets experienced some skin irritation at the injection sites; rotating the locations of the injections minimized this. Oral administration potentially provides longer periods of exposure; however, peak levels are not as high as those that follow parenteral administration [
48]. We also observed that some MgSO
4-treated ferrets began gagging and/or vomiting immediately after injections, which would have made repeated gavage difficult. In a clinical situation, tube feeding would negate this confounder. Perhaps the intervention was timed incorrectly. If the ventricular enlargement is too mild a therapeutic benefit might be difficult to detect or if the intervention is too late, no benefit might be possible. However, subgroup analysis of the 4 most severely affected ferrets in each group as well as the least severely affected still showed no significant differences between treatment groups.
We must also consider that the rodent experimental studies considered in the Introduction do not translate to larger animals. The majority documented only short-term benefit. In short term studies of brain hypoxic-ischemic damage in immature rat, sheep, and pig models, MgSO
4 has had inconsistent outcomes and results have been confounded by mild hypothermia; for this condition reviewers concluded “peripherally administered MgSO
4 is unlikely to be neuroprotective” [
49]. A meta-analysis of 8 randomized controlled trials with a total of 786 head-injured patients indicates that MgSO
4 has no significant improvement for mortality, but there is borderline improvement in the glasgow outcome scale [
50].
Authors’ contributions
DDC carried out the drug administration, behavioral work, dissections and histology, ELISAs, myelin enzyme activity assays, statistical analyses, and drafted the manuscript. DDC also assisted in the induction of hydrocephalus, MR imaging, and atomic absorption spectroscopy. ETB assisted in the induction of hydrocephalus, drug administration, behavioral work, MR imaging, and dissections. XM assisted with the MR imaging, behavioral tasks, ELISAs, myelin enzyme activity assays, and data collection. MDB conceived the study including its design and coordination, carried out the hydrocephalus induction, and guided writing of the manuscript. All authors read and approved the final manuscript.