Mechanism of metabolic stroke and spontaneous cerebral hemorrhage in glutaric aciduria type I
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Metabolic stroke is the rapid onset of lasting central neurological deficit associated with decompensation of an underlying metabolic disorder. Glutaric aciduria type I (GA1) is an inherited disorder of lysine and tryptophan metabolism presenting with metabolic stroke in infancy. The clinical presentation includes bilateral striatal necrosis and spontaneous subdural and retinal hemorrhages, which has been frequently misdiagnosed as non-accidental head trauma. The mechanisms underlying metabolic stroke and spontaneous hemorrhage are poorly understood.
Using a mouse model of GA1, we show that metabolic stroke progresses in the opposite sequence of ischemic stroke, with initial neuronal swelling and vacuole formation leading to cerebral capillary occlusion. Focal regions of cortical followed by striatal capillaries are occluded with shunting to larger non-exchange vessels leading to early filling and dilation of deep cerebral veins. Blood–brain barrier breakdown was associated with displacement of tight-junction protein Occludin.
Together the current findings illuminate the pathophysiology of metabolic stroke and vascular compromise in GA1, which may translate to other neurometabolic disorders presenting with stroke.
KeywordsMetabolic stroke Glutaric aciduria Blood–brain barrier Cerebral hemorrhage
Ischemic and hemorrhagic strokes have been extensively characterized and studied [1, 2]. A third type of stroke, known as metabolic stroke, begins with metabolic dysfunction and leads to a rapid onset of lasting focal brain lesions in the absence of large vessel rupture or occlusion [3, 4, 5]. The mechanism by which global metabolic dysfunction leads to focal brain injury in metabolic stroke is not well understood. Pure metabolic stroke is routinely reported in glutaric, isovaleric, methylmalonic and propionic acidurias . Additionally, the organic acidurias have been associated with spontaneous intracranial hemorrhage, suggesting a vascular component may contribute to brain injury in these disorders [6, 7]. The subdural and retinal hemorrhages frequently found in glutaric aciduria type I (GA1) may be mistaken for non-accidental head trauma, with severe legal and emotional consequences for families [7, 8]. The etiologic role of vascular pathology in metabolic stroke has not been previously elucidated.
GA1 provides a prototypical model for metabolic stroke as more than 90% of children with this disease will experience bilateral basal ganglia injury if not identified and treated pre-symptomatically [9, 10]. The disorder is caused by a deficiency of glutaryl-coenzyme-A dehydrogenase (EC 220.127.116.11; GCDH), inherited as an autosomal recessive condition . GCDH is required for complete oxidation of lysine, hydroxylysine and tryptophan. Affected individuals accumulate glutaric and 3-hydroxy-glutaric acids in the brain, which are believed to play a primary role in the pathophysiology of the disease. As one of the more common inherited metabolic disorders, GA1 affects 1:30,000 to 1:100,000 children worldwide [9, 12], with an increased frequency in genetically isolated populations such as Old Order Amish, Canadian Ojibway Cree natives, Irish Travelers, and native South Africans [10, 13, 14, 15]. Children with GA1 typically develop normally through early infancy, but then may experience an encephalopathic crisis associated with non-specific illness between 6 and 36-months of age . Prevention is critical as the encephalopathic crisis usually results in irreversible bilateral striatal injury with substantial morbidity including crippling dystonia, choreathetosis and shortened life span .
An animal model of GA1 encephalopathy was developed by providing GCDH-deficient (Gcdh−/−) mice with a high lysine or protein diet . Recent work with this model showed age-dependent susceptibility to acute brain injury similar to human GA1 that was associated with differences in the amount of brain lysine accumulation and subsequent conversion to glutaric acid in 4-week versus 8-week old Gcdh−/−mice . The young Gcdh−/− mice suffer seizures, paralysis, hemorrhages, and death within 3-6-days of lysine or protein diet exposure, with the acute accumulation of brain glutaric acid at levels found in human autopsy cases. Encephalopathy in Gcdh−/−mice was associated with energy deprivation, detected as depletion of α - ketoglutarate (α KG), ATP and phosphocreatine . Adult (> 8-week old) Gcdh−/− mice survive after consuming the same high lysine diet, but all develop bilateral striatal necrosis after 6-weeks .
Since the young Gcdh−/− mice develop hemorrhages and striatal injury similar to human GA1, this model provides the opportunity to investigate the specific vascular changes associated with an acute encephalopathic crisis. In the current study we first describe the neuropathology to guide our subsequent investigations, and then capture the evolution of the metabolic stroke including associated perfusion abnormalities. Additionally, we investigated the effect of metabolic compromise on the integrity of the blood–brain barrier (BBB), and potential changes in levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1-alpha (HIF-1 α). Our current findings detail the mechanism of brain injury in metabolic stroke and provide detailed evidence linking metabolic dysfunction to specific BBB abnormalities in Gcdh−/− mice. These data are likely translational to patients with GA1 as well as other neurometabolic disorders presenting with stroke.
All chemicals were purchased from Sigma (St Louis, MO, USA) unless otherwise specified.
Gcdh−/− mice and age-matched wild type (WT) or heterozygous (Gcdh−/+) controls, both of mixed C57Bl/6 J X 129SvEv background , were generated from homozygotes maintained at Penn State College of Medicine (Department of Comparative Medicine) All animal experiments were reviewed and approved in accordance with IACUC research guidelines set forth by Pennsylvania State University and the Society for Neuroscience Policy on the use of animals in neuroscience research as previously described .
Diets were purchased from Harland Teklad (Indianapolis, IN, USA). The standard diet was the Harland Teklad 2018 diet, which is 18% protein and provides 1% lysine by weight. The protein diet (TD.03637) (70% casein) contains 62% protein, which is 4.7% lysine by weight. The lysine diet (TD.04412) was prepared by adding free lysine to a standard diet to achieve 4.7% total lysine. This level of lysine is not toxic in normal animals [19, 20]. All special diet treated animals were evaluated daily for symptoms as previously described . In order to reduce the number of animals used for these experiments, the protein diet was used for all histology experiments . The lysine diet, which causes a slower onset of encephalopathy, was used for MRI experiments to avoid animals being too ill to be scanned and for long-term studies such as the BBB protein analysis.
Six 4-week old Gcdh−/−, 3 WT and 3 Gcdh−/+ mice were sacrificed before starting the diet (0 hour control). To follow the earliest pathologic events, 36 Gcdh−/−, 3 WT and 3 Gcdh−/+ mice were placed on a high protein diet at 4-weeks of age. Six Gcdh−/−mice were sacrificed every 12-hours after starting the diet and 3 WT and 3 Gcdh−/+ mice were sacrificed at 72-hours with the last 6 Gcdh−/− mice. A second group of 36 Gcdh−/−, 6 WT and 6 Gcdh−/+ mice were placed on the lysine diet at 4-weeks of age and processed similarly. All the above mice were perfusion fixed and processed for histology and electron microscopy. Four additional Gcdh−/− mice were immersion fixed to show in situ red blood cells and engorged vessels.
All mice were anesthetized with 100 mg/kg pentobarbital (i.p.), perfused with lactated Ringers (Baxter Deerfield, IL, USA) followed by 4% paraformaldehyde with 1% glutaraldehyde in 0.2 M cacodylate buffer for 15-minutes. For histology, brains were removed and post-fixed in 4% paraformaldehyde for 48-hours, and paraffin embedded. For electron microscopy, brains were removed and post-fixed for 48-hours in perfusion buffer fixative.
H & E slides were prepared from paraffin embedded brains. Sequential 10 μm thick coronal sections were made within 0.5 mm of the Bregma line for sections including striatum and between Bregma −1.5 and −2.0 for sections including hippocampus .
Brains were dissected into cortical, hippocampal or striatal blocks and embedded in Epon resin. Toluidine blue stained semithin Sections 1 μm thick were made from these blocks. Ultrathin sections of 90 nm were then cut from the same blocks and stained with uranyl acetate and lead citrate for transmission electron microscopic analysis using a Philips CM10 transmission electron microscope.
Immunohistochemistry and confocal microscopy
Glial fibrillary acidic protein (GFAP) and occludin (Occl) were detected using deparaffinized 10 μm thick coronal brains sections. Sections were blocked with normal serum and doubled labeled with polyclonal anti-GFAP (1:500) (Dako, Carpenteria, CA, USA) and monoclonal anti-Occl (1:200) (Zymed, South San Francisco, CA, USA). Additional slides were prepared with GFAP labeling alone. All incubations were in PBS with normal serum overnight at 4°C. Double or single labeled slides were washed separately 3 times each in PBS and incubated with goat anti-rabbit IgG coupled to Cy2 for GFAP and goat anti-mouse IgG coupled to Cy3 for Occl. Polyclonal GFAP alone was detected with goat anti-rabbit coupled to horseradish peroxidase (all secondary antibodies diluted 1:2000; Jackson ImmunoResearch, West Grove, PA, USA). All slides were counterstained with 4’,6’-Diamidino-2-phenylindole (DAPI) 0.1 μg/ml in PBS for 5 minutes. Confocal microscopy was performed using a Leica TCS SP2 AOBS confocal microscope (Leica Microsystems Wetzlar, Germany).
Capillaries were counted under 10× magnification of 1 μm thick semithin sections prepared as above. Coronal sections were examined from cortex, bregma zero; hippocampus, bregma-2.30 mm; and striatum, bregma zero according to ‘The Mouse Brain in Stereotaxic Coordinates’ . Total capillaries were counted within a 300 μm × 300 μm square section centered within the tissue sample. Capillaries were identified by the presence of at least one endothelial cell lining the lumen with identifiable nucleus and the correct size of 3–5 μm . Occluded capillaries were counted as collapsed or with stasis, showing no patent lumen.
Evans blue perfusion
Gcdh−/−, WT and Gcdh−/+ mice were placed on the normal or high protein diet for up to 72-hours (n = 5 per diet Gcdh−/−, n = 3 per diet WT or Gcdh−/+). Each animal was anesthetized as above and then perfused through the heart with 5% solution of Evans blue in normal saline for 30-seconds. Animals were sacrificed and brains were immersion fixed inside the skull for 48-hours in 4% paraformaldehyde. Brains were examined in whole and then sectioned under dissecting microscope. Additional control animals were used with brief (2-3-seconds) Evans blue injection followed by complete dissection to reliably differentiate arterial from venous structures. Evans blue concentration was measured in brain samples using spectrophotometry and corrected for protein content as previously described .
Western blot analysis
Brain protein extracts from mice from each treatment group (20 μg each as determined by Bio-Rad protein assay, Hercules, CA, USA) were loaded on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes, blocked in PBS-Tween 20 (0.1%) containing 5% non-fat dry milk and 0.1% BSA for 1 h at 4°C and incubated with monoclonal antibodies against Occludin, ZO-1 (both from Zymed, South San Francisco, CA, USA), Hypoxia inducible factor 1 alpha (HIF-1α) (RD System, Abingdon, England), vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti phospho-occludin at Ser 490  overnight at 4°C. Blots were washed with PBS-Tween 20 (0.1%) and incubated with horseradish peroxidase conjugated anti-mouse IgG (1:10,000) for 2 h at 4°C. Immunoreactive bands were visualized using an enhanced chemiluminiscence system (ECL; Amersham Biosciences). Densitometric analysis of immunoreactive bands was performed by using ImageQuant 5.2 software (Amersham Biosciences) and results were expressed as percentage of control (WT standard diet). As a loading control we reprobed the blots with anti-actin (Sigma-Aldrich) in order to control for small variability in the gel loading.
MRI angiography and perfusion
Magnetic resonance (MR) angiography and perfusion was performed on a 7.0 T Bruker system using a 2 mm birdcage coil. Gcdh−/− and WT mice were imaged at 0, 36, 72 and 96-hours following the start of lysine diet (N = 11 Gcdh−/−, N = 4 WT). Prior to imaging mice were anesthetized with 3% isoflurane, adjusted during the imaging to 1–1.5% in order to maintain a constant breathing rate of 40 bpm. Arterial spin labeling (ASL) was used to acquire a single-slice perfusion-weighted image at the level of the striatum (TR/TE = 2000/12 ms, NAX = 2, 1.06 mm slice thickness, 2082 μm2 in-plane resolution). A RARE sequence with multiple TR times (100-5000 ms), same slice position and resolution, was used to calculate T1 values. Perfusion values were calculated on a pixel by-pixel basis using NIH Image J (NIH; rsbweb.nih.gov/ij/) and MRI analysis calculator, a plug-in written by Karl Schmidt (NIH; rsbweb.nih.gov/ij/plugins/mri-analysis.html). MR angiography was done using a 3D gradient recalled echo sequence (TR/TE = 19/3 ms or TR/TE = 100/3 ms in order to allow slower moving blood to be visualized, 1173 μm3 voxel size, NAX = 1), and maximum intensity projection for image reconstruction.
Normally distributed data sets were analyzed by t-test, ANOVA or ANOVA with repeated measures with Fisher LSD or Holm-Sidak post-hoc test. Kruskal-Wallis one-way analysis of variance on ranks was performed with Student-Neuman-Keuls post hoc test on samples that were not normally distributed. Sigma Stat software (Jandel Scientific, San Rafael, CA) was used for analysis. All p-values less than 0.05 were considered statistically significant.
Venous congestion and hemorrhage
Microscopic inspection of perfusion fixed brain from Gcdh−/− mice on the protein diet showed evidence of hemorrhage (Figure 1f) and BBB compromise as red blood cells were found outside the endothelial cell layer (Figure 1g). Additional immersion fixed brain sections showed engorgement of larger non-exchange vessels (Figure 1h and i). Together these pathological changes suggest early filling of the venous system resulting in increased venous pressure, BBB breakdown and hemorrhage. Similar findings associated with venous dilation and engorgement have been reported in arteriovenous malformations where shunting of non-exchange vessels results in early filling of venous structures [25, 26].
Microscopic and ultrastructural changes
Ischemic changes in different brain regions
Percent occluded capillary vessels
0% ± 5%
75% ± 10%
45% ± 5%
12% ± 2%
0% ± 5%
25% ± 5%
80% ± 15%
40% ± 10%
0% ± 5%
0% ± 5%
50% ± 8%
90% ± 10%
Compromised capillary bed perfusion and increased blood–brain barrier permeability
In order to quantify these changes, we measured Evans blue concentration in perfused brain sections spectrophotometrically. Evans blue content did not vary significantly between Gcdh−/+ controls with and without the protein diet. Therefore, concentrations are reported as percentage of control after 72-hours of protein diet exposure (Figure 5, bar graph). Both Gcdh−/− cortex and cerebellum showed increased signal compared to control before protein diet exposure. This difference is likely associated with an increased baseline permeability of Gcdh−/− brain vessels. Gcdh−/− cortex, striatum and hippocampus all showed decreased signal compared to control consistent with occluded vessels and lack of capillary bed filling in Gcdh−/− brain 36-hours after protein diet exposure (Figure 5). After 48 hours of protein exposure, increased signal was found in all brain sections compared to control, consistent with increased BBB permeability after initial ischemia in these brain regions.
MRI angiography and perfusion
MR angiography showed reduced blood flow as a complete loss of signal from cerebral vessels between 72-96-hours after lysine diet exposure in Gcdh−/− mice. Figure 6b shows representative MR angiogram in WT and Gcdh−/− mice on a standard diet, and Gcdh−/− mouse after 96-hours of lysine diet. When an extended acquisition time (TR = 100 ms) was used to maximize the inflow signal and allow for slower blood flow to be visualized, only the posterior circulation including basilar and posterior cerebral arteries were identified (Figure 6b, far right). Taken together these results suggest a bilateral, frontal greater than posterior deficit in cerebral arterial flow associated with lysine diet exposure in Gcdh−/− mice. These findings are consistent with our Evans blue studies and previous findings in human GA1 showing decreased perfusion during encephalopathic crisis .
Molecular basis of blood–brain barrier weakness
Recent work by Murakami and colleagues  demonstrates a central role for occludin phosphorylation and trafficking away from the BBB in VEGF mediated BBB permeability. Based on the disruption of occludin localization at the BBB in Gcdh−/− mice, we investigated both VEGF levels and phosphorylation status of occludin in control and Gcdh−/− mice with and without lysine diet (Figure 7 and Additional file 1: Figure S1). VEGF, at baseline, was significantly increased in the cortex of Gcdh−/− mice compared to Gcdh−/+ control (Figure 7). After 36-hours of lysine diet exposure VEGF was further increased in the cortex. Preliminary results showed an increase in the phosphorylation status of occludin compared to control (Additional file 1: Figure S1). VEGF was also found to be elevated in the striatum after 36-hours, providing additional support for a role of VEGF in the abnormal localization of occludin (Figure 7) and increased vascular permeability (Figure 5). These data are consistent with incomplete occludin localization at the BBB in Gcdh−/− mice and previous studies that showed increased expression of VEGF in Gcdh−/− mouse brain . Testing for changes in hypoxia inducible factor 1 alpha (HIF1α) were inconclusive (data not shown). Increased VEGF has previously been associated with ischemic neurons , and is consistent with the evidence of tissue ischemia presented in Table 1 and Figures 5 and 6. Together these data suggest a mechanism of induced BBB weakness and permeability in GA1.
Cerebrovascular weakness has been frequently reported in GA1 as subdural and retinal hemorrhages, previously misdiagnosed as non-accidental head trauma [8, 10]. Similarly, the GA1 mouse model has consistently shown development of intracranial hemorrhages associated with encephalopathic crisis when exposed to protein or lysine diets that raise brain glutaric and 3-OH-glutaric acid levels . Previous in vitro studies have shown increased permeability of striatal but not cortical rat brain endothelial cell monolayers exposed to glutaric and 3-OH-glutaric acids . Similar work by Muhlhausen and colleagues showed disruption of human derived endothelial cells exposed to 3-OH-glutaric acid . The mechanism underlying a specific BBB weakness in GA1, however, has been elusive. Initial evidence that VEGF may play a role in these vascular anomalies came from microarray studies examining expression patterns in Gcdh−/− mouse brain that showed increased expression of VEGF . Recent data by Murakami and coworkers demonstrates the pivotal role of VEGF in mobilization of the tight-junction protein occludin away from the BBB . These data are consistent with our current findings that VEGF is increased and occludin is partially disrupted at baseline in the brain of Gcdh−/− mice and may account as a possible mechanism of BBB weakness in GA1.
Recent studies have shown that the interaction between HIF1 α and VEGF can be modulated by the availability of α KG . In light of our previous findings that α KG levels are depleted in the brain of Gcdh−/− mice, we can now propose a mechanism to explain how metabolic dysfunction causes specific BBB weakness in GA1 . As diagramed in Figure 8, production of glutaric acid in the brain is associated with depletion of α KG, which correlates with decreased glutamate and γ -aminobutyric acid (GABA) levels that are dependent on available α KG [17, 37]. Decreased α KG also leads to increased HIF1 α , which induces VEGF, already increased at baseline and exacerbated further during encephalopathy. VEGF stimulates phosphorylation and mobilization of occludin, which allows for increased permeability and loss of integrity of the BBB . In the context of a failing BBB, the increased pressure associated with shunting to non-exchange vessels during encephalopathy likely accounts for hemorrhages in GA1 [4, 28].
Striatal injury is the hallmark of neuropathology in GA1 and other disorders presenting with metabolic stroke [5, 38, 39]. Extrastriatal neuropathology involving the cortical grey and white matter are also reported, but less recognized and studied [40, 41]. Recent work by Harting and colleagues follows MRI changes in pre-symptomatic GA1 patients and shows delay in early frontotemporal cortical development that is already present at birth . In the majority of treated patients, this frontotemporal underdevelopment was shown to normalize by 4-years of age . In contrast, patients that experience encephalopathy are more likely to show frontotemporal atrophy and spongiform degeneration of white matter tracts [40, 41, 42]. Our current findings also show cortical involvement and parallel findings in GA1 patients include evidence of decreased perfusion and glucose uptake in frontocortical regions [4, 28, 40]. The GA1 mouse model may provide the opportunity to further study and elucidate specific differences between cortical and striatal susceptibility.
In both mouse and human GA1 encephalopathy, the cortex shows transient and more progressive degeneration while the striatum is injured more rapidly and completely . Similar findings were shown to be associated with the brain regions most affected by BBB breakdown in models of transient middle cerebral artery occlusion as well as metabolic inhibition [43, 44]. Striatal degeneration is the most consistent neuropathological finding in GA1 , however, our current findings and other models of ischemic and metabolic stroke show that cortical involvement typically precedes and exacerbates striatal damage [44, 45]. Indeed, striatal injury can be alleviated by removal of cortical projections [45, 46, 47]. Consistent with our current and previous findings, cortical neurons are more likely to recover as striatal neurons are further compromised paralleling the level of BBB damage [16, 48, 49]. Striatal neurons are especially at risk because of the high concentration of N-methyl-D-aspartate (NMDA) receptors and cortico-striatal glutamatergic projections as well as specific BBB vulnerability [16, 50]. Previous studies have shown the selective vulnerability of the striatum to uncontrolled excitatory amino acid exposure causing excitotoxicity . We and others have shown the specific vulnerability of the striatal BBB to metabolic and toxic insult as well as transient ischemic stroke [16, 44, 49]. Taken together, cortical insult and BBB compromise could account for selective striatal vulnerability. BBB compromise, as seen in this model, allows increased permeability of excitatory amino acids, which can lead to both acute and chronic neuronal injury with progressive degeneration seen in both mouse and human GA1. This mechanism could be in part responsible for bilateral striatal necrosis that develops over 6-weeks in adult Gcdh−/− mice surviving on a high lysine diet where BBB occludin is undetectable .
In the current study we have characterized metabolic stroke in a mouse model of GA1 showing initial neuronal swelling with secondary ischemia associated with impingement of brain capillaries. Capillary occlusion leads to shunting of blood to non-exchange vessels with early filling and dilation of the venous system. Loss of the tight-junction protein, occludin, is shown as an intrinsic weakness of the BBB exacerbated by metabolic encephalopathy. Together these findings may account for the intracranial hemorrhages frequently encountered in Gcdh−/− mice, and GA1 patients, and suggest potential new targets for preventive strategies. Previous work by Antonetti and colleagues showed hydrocortisone treatment of endothelial cells in vitro was associated with increased occludin localization at tight-junction barriers and decreased permeability . These findings suggest a potential role for hydrocortisone treatment to improve BBB integrity in GA1.
This work is dedicated to the memory of Michael Metil, whose persistent smile and dedicated parents taught us the importance of collaboration in the name of preventing further injury from GA1. We thank the Laverty and Oxford Foundations, the International Organization of Glutaric Aciduria, and the Maple syrup urine disease family support group as well as NINDS F32 Fellowship award, NS581642, to JL for support of this work.
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