Pontocerebellar hypoplasia type 2: a neuropathological update

Pontocerebellar hypoplasia type 2 (PCH-2; MIM 277470), an autosomal recessive neurodegeneration with fetal onset, was studied in six autopsies with ages at death ranging between 1 and 22 years. Three patients were distantly related. A case of olivopontocerebellar hypoplasia (OPCH; MIM 225753) was studied for comparison. Typical findings are: short cerebellar folia with poor branching (“hypoplasia”), relative sparing of the vermis, sharply demarcated areas of full thickness loss of cerebellar cortex probably resulting from regression at an early stage of development, segmental loss of dentate nuclei with preserved islands and reactive changes, segmental loss in the inferior olivary nucleus with reactive changes, loss of ventral pontine nuclei with near absence of transverse pontine fibers and sparing of spinal anterior horn cells. Variable findings are: cystic cerebellar degeneration, found in two, with vascular changes limited to the cerebellum in one. Comparison to olivopontocerebellar hypoplasia (OPCH) strongly suggests a continuum of pathology between this disorder and PCH-2. Immunohistochemical evaluation of the endoplasmic reticulum stress response is negative. We conclude that the neuropathological findings in PCH-2 are sufficiently specific to enable an unequivocal diagnosis based on neuropathology.
Introduction
The term pontocerebellar hypoplasia (PCH) is applied to a group of autosomal recessively inherited neurodegenerations with prenatal onset. The distinguishing attribute of this group is the presence of combined pontocerebellar hypoplasia and atrophy present at the time of birth.
Supratentorial structures are affected, though less prominently. The group has not been clarified genetically or biochemically. An initial proposal for classification [6] is based on two types in which type 1 (PCH-1) associates PCH with spinal anterior horn cell degeneration [14, 33], while type 2 (PCH-2) associates PCH with clinical extrapyramidal involvement and absence of anterior horn degeneration [5–7, 37, 44]. Early detailed neuropathological reports on PCH [9, 10, 26] in retrospect may have been cases of PCH-2, but cannot be matched with the customary classification because of lack of clinical details. Neuropathological finding in PCH-2 [5, 7, 37, 41] is microencephaly with severe cerebellar and ventral pontine hypoplasia. Typical pathological findings are: (1) subtotal loss of ventral pontine neurons and transverse pontine fibers with preservation of long fiber tracts; (2) atrophy and hypoplasia of the cerebellar hemispheres with relative sparing of the vermis and flocculi, variable loss of Purkinje cells and internal granule cells and reduction in folial length; (3) patchy loss of cerebellar dentate neurons, with the remaining dentate neurons grouped in “islands”; (4) loss of neurons in the inferior olivary nucleus with the winding pattern essentially spared; (5) absence of the medullary arcuate nuclei. Findings in PCH-1 are essentially similar, but preservation of spinal anterior horn cells distinguishes PCH-2 from PCH-1 [14]. Microscopic supratentorial findings in PCH-2 are non-specific with neuronal loss in various compartments, including the cerebral cortex, while myelin and myelination remain unaffected. Ultrastructural findings in a single reported neocortical biopsy are progressive neuronal loss in all layers and a peculiar degeneration in neurons in all layers that appear to start with patches of darkened endoplasmic reticulum [5]. Diagnosis during life is based on a combination of findings on MRI (Fig. 1), a profile of clinical neurological deficits that includes severe cognitive delay, swallowing disturbance and chorea/dystonia (less often, spasticity), exclusion of metabolic and chromosomal disorders with a similar MRI pattern and a family history compatible with autosomal recessive inheritance. In typical cases, behavioral and motor development is almost stagnant from the beginning, with onset of chorea/dystonia during the first year and progressive microcephaly [7, 44]. Differential diagnosis requires exclusion of glycosylation disorders, especially congenital disorder of glycosylation type 1A (CDG1A) [2, 21, 23] and Muscle-Eye-Brain disease [16, 29], sequelae of extreme prematurity [28] and chromosomal disorders [4]. Overlapping neuropathological features exist between PCH-2 and olivopontocerebellar hypoplasia (OPCH), a more severe disorder with an essentially similar combination of hypoplasia and degeneration affecting the hindbrain [3, 11, 18, 35]. Autosomal recessive inheritance in OPCH is suggested by its recurrence pattern in families. Clinical findings are variable, but include such indicators of prenatal onset as polyhydramnios and contractures. Its main structural features are severe cerebellar hypoplasia with emphasis on the hemispheres, absence of neurons in the ventral pons, subtotal absence of cerebellar dentate nuclei, diminished or absent olivary winding and absence of spinal anterior horn involvement. In a recent PCH classification, the original types 1 and 2 have remained identical, while olivopontocerebellar hypoplasia has become type 4, PCH-4 [36]. Type 3, with optic atrophy as its main distinguishing clinical feature [38], has not been studied neuropathologically yet, while type 5 until now is represented by a single family. The number of papers addressing the neuropathology of PCH-2 with sufficient clinical detail to support a type 2 classification is limited [5, 37, 41] and results of immunohistochemistry have not been reported yet. The aim of the present report is to provide a range of pathological findings based on a series of six autopsies with ages at death varying between 15 months and 22 years and to provide an update on neurodegenerative features using more specific staining techniques. Special attention is given to variability in cerebellar degeneration, degenerative changes in pons and lower brainstem and the types of astroglial and microglial reaction. Three patients (no. 4, 5, 6) originate from an area with a high degree of intermarriage and are related by genealogy. A case of olivopontocerebellar hypoplasia (PCH-4) is included for comparison of its neuropathology to PCH-2.Fig. 1MRI of patient 6 at 2 months. a Midsagittal inversion recovery sequence: flat aspect of ventral pons and moderate vermal hypoplasia. b Coronal T2-weighted image: severe hypoplasia of the cerebellar hemispheres (arrows) together filling only a fraction of the posterior fossa
Materials and methods
Standard autopsies were performed on all patients mentioned except patient 1 who underwent a coroner’s autopsy with only the cerebellum and brainstem made available for the present study (Table 1).
Table 1Clinical profiles, macroscopic and principal microscopic findingsPatient no.1234a5a6a7SexFemaleMaleMaleMaleMaleFemaleMaleType of PCHPCH-2PCH-2PCH-2PCH-2PCH-2PCH-2OPCHParents, relatedNoNoYesYesYesUncertainYesNeonatal periodPoor swallowingPoor swallowing, seizureUnrestSeizurePoor swallowingPoor swallowing, unrestAbsent spontaneous respiration, tremor/clonusHead circumference at birth−1 SD−2 SD−2 SDMicrocephaly progressive after birth++++++Motor development after birthNo motor control, some visual pursuit Minimal head control, some visual pursuitMinimal head control, some visual pursuitNo motor control, some visual pursuitNo motor control, no visual pursuitNo motor control, no visual pursuitChorea/dystonia++++Spasticity+Forced flexion of extremitiesSeizures++++++−Previous publicationPatient 9, [7]Patient 11, [7]Patient 7, [7]Patient 3, [7]–––Age at death1 year 2 months12 years22 years 10 months15 years 6 months2 years 4 months3 years 7 months2 daysCause of deathCot deathHyperthermiaCachexiaPneumoniaPneumoniaHyperthermia, rhabdomyolysisAbsent spontaneous respirationTotal brain weight (g)570720 1,110After fixation, 687660660After fixation, 172Control brain weight g ± SD*940 ± 120b1440 ± 10b1440 ± 20b1410 ± 10b1120 ± 200b1090 ± 230b433 ± 57cWeight of cerebellum + brainstem3216–––12–Cerebellar cortexSparse segmental lesions in hemispheres; diffuse loss of P- and granule cells Sparse segmental lesions in vermis and hemispheres; diffuse loss of all P- and granule cellsNear total destruction, only GFAP positive outlines remainingSegmental lesions in hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsMajor (dorsal) part of vermis and hemispheres “denuded”, sparing of flocculus and nodulusDentate nucleusIslandsIislandsNo remnantsIslandsIslandsIslandsIslandsCerebellar cysts−−+−+−−Inferior olivary nucleusReduced folding; segmental lesionsReduced folding; segmental lesionsTotal loss of structureReduced folding; segmental lesionsReduced folding; segmental lesionsReduced folding; segmental lesionsNo foldingVentral ponsLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersaRelated by common ancestry (Fig. 2)bReference for fresh brain weights [12]cReference for fixed brain weights [15]
Tissue preparation and immunocytochemistry
Formalin-fixed, paraffin-embedded tissue was sectioned at 6 μm and mounted on organosilane-coated slides (SIGMA, St Louis, MO, USA). Representative sections of all specimens were processed for immunocytochemical reactions to synaptophysin (polyclonal rabbit, DAKO, 1:200), microtubule-associated protein (MAP2; polyclonal rabbit, Sigma, 1:2,000), calbindin (mouse clone CL-300, Sigma, 1:400), SMI31 (Sternberger Monocl., IgG 1, 1:20,000), phosphorylated neurofilament (NF; mAb clone 2F11, DAKO, 1:500), myelin basic proteïn (MBP; polyclonal, Neomarkers/RB-1460-A, 1:400), vimentin (mouse clone V9, DAKO, Denmark, 1:1,000), glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Denmark, 1:4,000), (HLA)-DP, DQ, DR (mouse clone CR3/43, DAKO Glostrup), HLA-DR (mouse clone Tal1b5, Sigma, USA, 1:200), CD68 (KP1 clone, Novocastra), caspase-3 (polyclonal, 1:100 dilution, Cell Signalling Technology, Beverly, MA, USA), ubiquitine (polyclonal, DAKO/Z458, 1:1,600), α-synuclein (polyclonal, Affiniti/SA 3400, 1:2,000), BiP/GrP/78 [polyclonal rabbit, 1:100 dilution Santa Cruz Biotechnology (SCBT), Santa Cruz, CA, USA], phosphorylated (p)PERK (pThr981, polyclonal rabbit, 1:800 dilution, SCBT) and phosphorylated (p)eIF2α (pSer51, polyclonal rabbit, 1:500 dilution, Sigma).
Paraffin sections were routinely stained with HE, LFB and Nissl. Cerebellar sections were silver stained according to Bielschowsky. Immunocytochemistry was carried out on paraffin-embedded tissue as previously described [2, 19]. The sections were incubated for 1 h at room temperature followed by incubation at 4°C overnight with primary antibodies. Single-label immunocytochemistry was performed using avidin-biotin peroxidase method. Chromogen 3,3-diaminobenzidine or 3-amino-9-ethyl-carbazole (AEC, Zymed, San Francisco, CA) was used and nuclei were stained with hematoxylin. Sections incubated without the primary Ab, with preimmune sera were essentially blank. For double labeling (with ubiquitin and HLA-DR), sections were incubated for 2 h at RT with Alexa Fluor® 568 and Alexa Fluor® 488 (anti-rabbit IgG, anti-mouse IgG; Molecular probes, Eugene, USA). The sections were then analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA; MRC1024) equipped with an argon-ion laser.
Results
Details of each patient are given in Table 1. Figure 2 shows the pedigree, which links patients 4, 5 and 6 to a common ancestry. Parents of patients 4 and 5 are consanguineous. The three parental couples originate from the Dutch community in Volendam, which has a high degree of consanguinity. They link genetically to one or two ancestral couples: I, 1 × I, 2 and III, 4 × III, 5 born in the eighteenth century. A genealogical link between these ancestral couples, who carry identical family names, is suggested but yet unproven.
Fig. 2Pedigree linking patients 4, 5 and 6 in the present report; IX, 1 and IX, 3 were reported before [5]
Cerebellar cortex
The cerebellar hemispheres are severely affected in all. On macroscopic examination, all major lobes are reduced in width and dorsoventrally flattened. Size was best preserved in the transverse plane. This asymmetric diminution in size and the relative sparing of the vermis lend the cerebella a butterfly or batwing aspect (Fig. 1b). Four of the six cerebella (cases 2, 4–6) are sectioned perpendicular to the folia with the line of the sectioning running from the culmen to the posterior pole. These sections include the flocculus. In two cases (1, 3), transverse sections of hemispheres and vermis are prepared. Low magnification (Fig. 3) shows the diminished size of the cerebellar hemispheres, folial shortening and diminished number of folial branches compared to a control (Fig. 3a, b). In the most affected case (Fig. 3g, h), no branches are seen at all, while in a less affected case folial branches are reduced to two or less (Fig. 3e, f). The other cerebella displayed regular folial branching (Fig. 3c, d), though less in quantity than the control specimen (Fig. 3a, b).
Fig. 3Hypoplastic folia. Stains, synaptophysin. Sagittal sections of cerebellar hemispheres are seen on the left and detailed views on the right. Stunted folial growth with decreased number of folial branches. a, b Male, 8 years, with accidental death and normal findings on autopsy (control); c, d case 4; e, f case 6; g, h case 5. Marker bars on the left 1 cm, and on the right 1 mm
The vermis is fully developed in all cases with the individual lobules well developed in size and branching pattern. Sagittal sections of the vermis, available in four, show some inequality in thickness of the white matter cores of the lobules, with the declive–folium–tuber complex the best preserved part in three (cases 2, 4, 5) and the pyramis in one (case 5; Fig. 4).
Fig. 4Vermis cerebelli, case 5. Stains, a HE, b GFAP, c neurofilament protein, d vimentin. a–c Adjacent sections with circumscript segments of loss of all layers and glial reaction. Arrows in (a) point to circumscript areas of lost cortex. d Sharply demarcated foci and replacing bands of astrocytes possibly modified Bergmann cells. Marker bar in (d) = 500 μm
On microscopy, regressive changes are present in all, with the vermis relatively spared. In the vermis, diffuse but moderate loss of Purkinje cells, internal granule and folial white matter is seen in all lobules. Two cases present characteristic sharply delimited foci with full thickness loss of vermal cerebellar cortex with astroglial replacement (2, 5; Fig. 4).
The hemispheric cerebellar cortex is affected in all cases by variable loss of Purkinje cells, thinning of the granule cell layer and inhomogeneous loss of myelinated fibers within folia and central white matter. The remaining axons are well myelinated and stain for SMI 131 and MBP. In addition to diffuse cortical changes, sharply delimited areas of up to 2 mm in width with loss of all cortical neurons are found in 5/6 cases (1, 2, 4, 5, 6). These areas locate mostly, but not exclusively, to stretches of cortex connecting adjacent folia. The number of these areas varies between two per whole cerebellar hemispheric section (1, 2) to ten or more (5, 6; Fig. 5). In one case (3), representing the oldest surviving patient, destruction of the whole cerebellum is almost complete with subtotal loss of all neuronal elements (Fig. 10a). Even in this case, patches of whole thickness loss of cortex alternate with segments of cortex where gliosis still preserves the original frame of cortical structure (Fig. 10a). In all cases, the flocculus belongs to the relatively spared areas of the hemispheric cortex. In one case, the sparing of the flocculus stands out in sharp contrast to the rest of the hemispheric cortex (Fig. 6).
Fig. 5Cerebellar hemispheric cortex with segmental lesions. Stains, a, h synaptophysin, b–d vimentin, e, g, i GFAP, f MAP-2. Case 6: opposing arrows mark lesions with loss of all cortical layers (a) and glial replacement (b). c, d Case 5: segment of whole thickness loss of cortex is replaced by a single layer of evenly spaced astrocytes, their topography compatible with modified Bergmann cells. e, f, g Case 4: segmental loss of cortex with glial replacement marked by opposing arrows; in (f) higher magnification shows a group of remaining isolated granule cells. h, i Case 2: segmental loss of cortex on top of a folium in moderately hypoplastic cortex. Marker bars: a–c, e, g–i = 0.5 mm, d = 0.25 mm, f = 50 μmFig. 6Flocculus. Cerebellar hemispheric cortex, case 2, synaptophysin. Floccular cortex shows broader folial cores and denser granule cell population than other parts of the cortex
Dentate nucleus
The dentate nucleus is affected in all cases by major loss of dentate neurons and by residual cells being grouped in “islands” (Figs. 7, 8). “Islands” as well as individual neurons are surrounded by whorls of nerve fibers. Residual neurons and the proximal parts of their dendrites stain positive for MAP-2 and synaptophysin. Synaptophysin staining is abundant on neurons and proximal dendrites signifying retained network activity in surviving neurons. A peculiar vacuolization surrounds individual dentate neurons (Fig. 8a, b). A proportion of these vacuoles stain positive for calbindin, suggestive of swollen axon terminals (Fig. 8c, d). Cerebellar white matter surrounding the dentate nucleus on the inside and outside shows thickened axons with diameters up to 12 μm (Fig. 8). The other internal cerebellar nuclei could not be identified with certainty.
Fig. 7Dentate nucleus, case 6. MAP-2. Loss of normal outline with remaining dentate neurons clustered in “islands”. Marker bar = 1 mmFig. 8Dentate nucleus, details. Stains: a MAP-2, b MBP, c, d calbindin, e synaptophysin, f SMI31. a Case 6: fine vacuolation surrounding neurons. b Case 1: myelinated fibers separate individual neurons and their surrounding vacuoles. c, d Cases 6, 1: some vacuoles are decorated by antibody; e Case 2: concentration of vesicles (dots) indicates high synaptic density on soma and dendrites of surviving neuron. f Case 1: thickened axons in hilus of dentate nucleus. Marker bars = 100 μm
Cystic degeneration of the cerebellum
In two cases, destruction resulted in cyst formation (cases 3, 5). MR investigation shows cystic change in the cerebellar hemispheres of patient 5 at the age of 1 month (Fig. 9b ). On autopsy the cysts are collapsed, and can only be retrieved by serial sectioning. The border of the cyst is lined by reactive astrocytes and macrophages (Fig. 9a, c). In case 3, cyst formation compounds the subtotal loss of cortex, dentate nuclei and fiber tracts. A remarkable involvement of small sized vessels, not present in the other cases, consists of intimal proliferation and splitting of the elastica interna. This abnormality is only present in the cerebellum and its surrounding arachnoid vessels (Fig. 10). Electron microscopy fails to show storage material or enlarged lysosomes (not shown). Double labeling for ubiquitin and HLA-DR shows that ubiquitin is mainly present in the vessel walls, and only in minor part associated with macrophages (HLA-DR; Fig. 10c). Vascular abnormalities are absent in other parts of the brain and in internal organs, including the kidneys.
Fig. 9Cerebellar cyst, case 5. a Vimentin, c HLA-DR. a Collapsed cyst seamed by astroglial reaction. b T1-weighted axial image of posterior fossa of patient at 1 month age shows the cysts in expanded state. c Macrophage reaction on the inside of the cystFig. 10Regressive changes, case 3. Stains: a GFAP, b HLA-DR, c double labeling for HLA-DR (green) and ubiquitine (red), confocal laser microscopy, d ubiquitin. a Transverse cerebellar section with cortical and subcortical gliosis, loss of the subcortical and central cerebellar structure, in part cystic, broken cortical lining in places suggest segmental lesions similar to other cases before ultimate decay; b abundance of reactive macrophages, predominantly in and surrounding blood vessels with thickened vessel walls; c ubiquitin is mostly restricted to vessel wall and macrophages are partly positive for ubiquitin; d neuron of nucleus magnus raphes (pontine tegmentum) with cytoplasmic inclusion and positivity extending into the neurite. Marker bars: b = 200 μm, c = 50 μm
Inferior olivary nuclei
The inferior olivary nucleus and the dorsal and medial accessory olivary nuclei are identified in each case. Mature folding of the inferior olivary nucleus is present in the lateral leaf and reduced in the medial leaf in one case (3; Fig. 11). In all others, folding is reduced in both leaves, the medial leaf being more affected than the lateral. Discontinuous loss of parts of the nerve cell band is noted in each case, best shown by MAP-2 staining (Fig. 11). In one case (3) nearly all olivary neurons, including the accessory olivary nuclei, are lost. Part of this loss is mirrored by vimentin positive astrocytes forming a ghost image of the lost neuronal framework (Fig. 11c, d). Macrophages are present within and around the vanished structure, but not in a topographically distinct pattern. In all cases, internal and external olivary fibers are reduced or lost entirely.
Fig. 11Inferior olivary nucleus. Stains: a–c MAP-2, d vimentin. case 6 and 5: segmental loss of inferior olivary nucleus with intact dorsal (a) and medial (b) accessory nuclei. c Case 3. Complete loss of nucleus, including accessory nuclei, only sparse isolated neurons remaining, indicated by arrowheads; inset shows magnified solitary olivary neuron. d Case 3. Outline of lost lateral leaf reveals mature folding. Marker bars = 0.5 mm
Pons
Ventral pons
Similar findings are present in all cases. Diminution in size of the pons to about half or less of its size in transverse diameter is entirely due to loss of its ventral compartment. The majority of ventral pontine neurons and transverse pontine fibers are lost with sparing of the long motor and sensory tracts. Prominent gliosis (Vim and GFA) and microglial activation are found in the affected region. Staining for synaptophysin and calbindin in the remaining neurons does not reveal specific patterns. Caspase and ubiquitin staining is negative in the few remaining neurons.
Pontine tegmentum
No abnormalities are encountered in the cranial nerve nuclei, raphe or central tegmental tract. Ubiquitin staining in the reticular nuclei, especially the nucleus raphes magnus in case 3, reveals numerous neurons with ubiquitin-positive cytoplasmic inclusions. Caspase-3 staining is negative in all cases.
Other findings in the brainstem
Total absence of the medullary arcuate nuclei is found in all cases. Widespread presence of lipopigment (lipofuscin) is observed in all cases, especially in larger neurons (dentate nuclei, olivary nuclei, pontine neurons, brainstem tegmentum and cerebral cortex), even in the youngest individuals. Staining for α-synuclein was negative in the brainstem as well as in the cerebellum in all cases.
Midbrain, thalamus and basal ganglia
Nucleus ruber has diminished size and cell density in case 3. This case contrasts with other cases by its subtotal destruction of the cerebellum. In all available cases (2–6), the caudate nuclei are macroscopically atrophic. There is a general increase of astrocytes and microglia throughout the basal ganglia and thalami. Myelination is normal. No specific abnormalities are found. In one case severe necrotic changes are found in the globus pallidus, representing acute postischemic damage.
Cerebral cortex
Macroscopic gyral atrophy is seen in three of five cortices available for study. The temporal lobes are affected in one (case 2), the frontal and temporal lobes in one (case 3) and all lobes in one (case 4). Myelination is normal in large sections (luxol fast blue), even in atrophic parts. Cortical development appears normal on routine staining. A diffuse increase of reactive astroglia and activated microglia is seen throughout the neocortex and subcortical and central white matter in all cases. Except for postischemic changes in one case (6), the hippocampal and dentate gyri are normal.
Olivopontocerebellar hypoplasia
Case 7: this male baby was born at 38 weeks ga. The amount of amniotic fluid was normal. Spontaneous respiration was absent. He had trismus, severe tremor/clonus and varus deformity of the legs. On autopsy, the supratentorial parts of the brain are small size, but of normal appearance, while the cerebellum and brainstem are disproportionately small.
The cerebellum is remarkable for its complete sparing of the nodulus and flocculus, its severe cortical depletion on the dorsal side and hypoplastic cortical development on the ventral side of the hemispheres (Fig. 12). Folial development of the neocerebellum is severely restricted with few folia on its dorsal parts. Microscopic examination of the flocculus and vermis is normal. Neocerebellar hemispheric cortex shows isomorphic gliosis through transformed Bergmann cells (Fig. 12b). MAP-2 staining shows some retained neuronal elements (Fig. 12c, d). The dentate nuclei are broken up into small islands that can only be retrieved by neuron-specific staining. This reveals a few small clusters with well-developed dentate neurons left in place (Fig. 12e, f). The inferior olivary nuclei have the form of horseshoes without folding, and with the segments of the nucleus missing on both sides (Fig. 13). The accessory olivary nuclei are spared. The ventral pons is severely underdeveloped with most of the pontine nuclei missing, dense gliosis and absence of CD68 staining. A diffuse astrocytic gliosis is found throughout the cerebral hemispheres, especially affecting the subcortical white matter and the fiber tracts of the striatum. The neocortex is unremarkable.
Fig. 12OPCH, case 7. Stains: a, e synaptophysin, b vimentin, c, d, f MAP-2. a Whole mount transverse section of cerebellum and medulla oblongata: denuded hemispheric cerebellar cortex and striking preservation of nodulus and flocculi. b Hemispheric cortex lined by a single row of fibrous astrocytes. c “Empty” cerebellar cortex, arrows indicate some remaining neurons. d Higher magnification of c, arrowhead indicates bipolar neuron, possibly basket cell; arrow indicates malpositioned Purkinje cell. e Overview of cerebellar hemisphere, arrows at residues of dentate nucleus. f Single residual island of dentate nucleus with well-developed neurons. Marker bars: b 200 mμ, c 1 mm, d 100 mμ, e 1 mm, f 100 mμFig. 13OPCH, case 7. Stain, synaptophysin. Medulla oblongata with olivary nuclei. Inferior olivary nucleus with complete lack of folding. Lateral and medial leaves with segmental defects. Medial accessory olive is preserved in its dorsal part and shows discontinuities in its ventral part. Marker bar 0.5 mm
Unfolded protein response
We previously observed morphological changes in the endoplasmic reticulum (ER) in a biopsy of a PCH-2 patient by electron microscopy. This led us to investigate whether the unfolded protein response (UPR)—a stress response of the ER [40]—is activated in neurons of PCH-2 patients. The UPR was studied in sections from the frontal cortex (5/6 patients) and the cerebellar hemispheres, which included the dentate nucleus (6/6 patients). As a marker of induction of the UPR, we used an antibody specific for the phosphorylated pancreatic ER kinase (pPERK), which is an ER stress transducer that is only phosphorylated during the UPR. Previously, we have used this method to show UPR activation in the brain of patients with Alzheimer’s disease [19] or Parkinson’s disease [20]. We find no immunopositive staining for pPERK in any of the patients, neither in the cortex, nor in the cerebellum. Eukaryotic initiation factor 2α (eIF2α) is the substrate of pPERK and using a peIF2α antibody, increased staining intensity is observed as compared to the control patient in the Purkinje cells of patients 2, 4 and 5. This is not found in patient 1 and severe degeneration in patients 3 and 6 precludes proper analysis. Apart from the fact that differences in intensities between pathological samples are not always readily interpretable, this does not provide evidence for UPR activation in the absence of pPERK reactivity, because other kinases can also phosphorylate eIF2α. The ER chaperone BiP is upregulated during the UPR, but in accordance with the pPERK data, no obvious differences in levels are observed. In summary, we find no evidence for activation of the UPR.
Discussion
Cerebellar cortex, dentate nucleus and olivary complex
The series of six PCH-2 cases presents developmental and regressive abnormalities of the cerebellar cortex, dentate nuclei and olivary nuclei. Cerebellar cortical findings range between short unbranched folia in the least-developed cerebellum (case 5) to shortened folia with a decreased number of branches. In the most affected cerebella, poorly developed folia alternate with flat stretches of surface that lack proper cortical lining. A similar observation is reported in PCH-1 cases [13, 14, 46], OPCH [35], a case classified as “neocerebellar hypoplasia with combined olivo-ponto-dentatal degeneration” [24], and PCH-2 with spasticity [42]. Reactive glial changes were reported in PCH-1 [46]. All “empty” segments of cortex in the present study have a similar appearance: a single evenly spaced row of transformed Bergmann cells, positively staining for vimentin. Synaptophysin and MAP-2 staining reveal the sparse presence of nerve fibers. This is the first time this patten is observed by immunohistochemistry. The earliest stages of cerebellar development are probably normal because they require the coordinate development and juxtaposition of two cell types: the Purkinje cells that arise by radial outward migration and the external granule cells that arise by tangential migration and enter the future cortex to become internal granule cells [39]. The microscopic structure of the “empty” segments betrays previous cortical development. The earliest estimate for the loss of cortex in these segments follows from the afolial stretches in patients 5 and 6 that represent a stage of cerebellar development prior to or coinciding with beginning folial outgrowth, around 25 weeks gestational age in the human fetus [39]. Cortical segmental loss, however, is not restricted to afolial segments, but is also seen in more developed hemispheric cortex (e.g., Fig. 5h, i) and mature vermis. This suggests a time frame for segmental loss of cerebellar cortex from incipient foliation to encompass late cortical development. It cannot be established presently whether segmental loss is a completed process or still active at the time of demise. No microscopic vascular changes are seen in the vicinity of segmental lesions and their distribution is not restricted to arterial boundary zones. Also, cerebellar vascular changes observed in one case (3) are not found in any of the other cases and therefore offer no explanation for segmental cortical lesions. The spectrum of changes in the inferior olivary nucleus in this series similarly encompasses early and late developments. In five out of six cases, the degree of folding is immature, while in the sixth case (case 3), a mature folding pattern of the nucleus can be gauged by observing the outline of the astroglial scar. According to one study in human fetal material [30], the mature form of the nucleus is reached with a 300 mm crown–heel length, i.e., 22–27 weeks. Segmental loss is observed in 5/6 cases in the olivary nucleus, and the sequence of progressive loss may have been similar in the remaining oldest case (case 3) with loss of the entire nucleus. Segmental lesions in the inferior olivary nucleus in PCH were reported in 1926 [26]. The consistency of this finding is shown in the present series using immunohistochemistry. The third structure with a segmental pattern of loss is the dentate nucleus. We have no evidence in this series of a normal outline of the dentate nucleus during any phase of its development. One previous report [32] however relates a glial scar linking the “islands” of the dentate in PCH.
We examined a case classified as OPCH and on comparison to the PCH-2 series found important similarities: (1) flat stretches of cerebellar cortex and ghosts of primitive folia carrying microscopic remnants of a pre-existent developed cortex, (2) dentate islands with well-developed neurons, (3) segmental loss in the inferior olivary nucleus, (4) sparing of nodulus and flocculus. These findings reveal a remarkable analogy to the segmental cortical lesions in PCH-2. Differences between PCH-2 and OPCH are: (1) the horseshoe appearance of the inferior olivary nucleus compared to the diminished folding in PCH-2 indicates earlier onset of the pathological process in OPCH, (2) the extent of afolial cerebellar cortex.
Regressive changes
In addition to developmental abnormalities, some findings indicate an ongoing process, such as cytoplasmic ubiquitin positivity in pontine tegmental neurons in case 3, the widespread presence of astrocytic and microglial reaction and the abundance of lipopigment, e.g., in cerebral cortical, cerebellar cortical, dentate and olivary neurons. Cyst formation in the cerebellar white matter (cases 3, 5) and vascular changes in the cerebellum (case 3) also signify a regressive element in PCH-2. A previously made observation on darkened stretches of endoplasmic reticulum in cortical neurons in a cortical biopsy [5] prompted us to investigate the ER stress response with negative results.
Comparison of PCH-2 to other neurodegenerative disorders
The nearest equivalent in neuropathological terms to PCH-2 is the group previously named olivo-ponto-cerebellar atrophy (OPCA), presently known as spinocerebellar ataxia (SCA). While olivary and/or dentate nucleus may be affected, none of the typical features, especially dentate fragmentation, segmental lesions of the cerebellar cortex and olivary nucleus is part of the pathological spectrum of these disorders [8, 25]. Pathological studies on autosomal recessive or X-linked central nervous system disorders with onset in childhood and predominant impact on the cerebellum include the Høyeraal-Hreidarsson syndrome [1, 22], progressive encephalopathy, hypsarrhythmia, optic atrophy (PEHO) [16], primary granular cell degeneration of the cerebellum [31], and infantile onset spinocerebellar ataxia with neuropathy (IOSCA) [27]. These disorders display cerebellar atrophy rather than hypoplasia, lack the segmental cortical anomalies, the characteristic fragmentation of the dentate and the segmental loss of the olivary nucleus described above with the possible exception of IOSCA in which segmental loss of the olivary nucleus is documented [27]. Neurometabolic disorders of glycosylation, especially congenital disorder of glycosylation (CDG) type 1A and the alpha-dystroglycanopathies manifest cerebellar hypoplasia, often in combination with pontine hypoplasia. Neuropathological findings in CDG1A do not include circumscript dentate, olivary or cerebellar cortical defects [2, 21]. Alpha-dystroglycanopathies combine a severe type of congenital muscular dystrophy with cerebral, cerebellar and brainstem malformation and deficiency of alpha-dystroglycan (alpha DG) [29]. Alpha DG deficiency in this group probably causes a tangential neuronal migration defect affecting migration from the rhombic lip into the cerebellum and ventral pons. The resulting pathology [17] causes heterotopia and impaired foliation in the cerebellum. Summarizing previous and present data on PCH-2, this disorder bears a number of features not encountered in other disorders with the exception of PCH-1, which appears to be closely related but for the involvement of spinal anterior horn cells in the latter.
Neural mechanisms
A question may be raised whether segmental lesions in the three nuclear systems, as observed in PCH-2, could be effected through a single mechanism. Cerebellar cortex, olivary nuclei and dentate nuclei are functionally linked through the climbing fiber system, which is topographically organized in sagittal modules each formed by a segment of the olivary nuclear complex, its collaterals to the cerebellar nuclei, including the dentate nucleus, and a strip of cerebellar cortex [45]. A sagittal parcelleation of the cerebellum in biochemically distinct regions, however develops independent of the olivo-cerebellar projection [43]. Lesions of the olivary complex are not known to cause segmental lesions in other parts of the climbing fiber system. Experimental administration of the drugs harmaline or ibogaine to rats results in tremor and loss of sagittal strips of vermis. The role of the olivary nucleus in this type of segmental damage is highlighted by prior chemical ablation of the inferior olivary nucleus by 3-acetyl pyridine, which prevents the segmental damage from occurring. Overexcitation of the olivary nucleus rather than primary loss is apparently required for this type of lesion to occur [34]. The type of lesion reported in rodents is reminescent of the segmental lesions in PCH-2. One argument in support of a possible role of the olivo-cerebellar projection in PCH-2 is the selectivity of the cerebellar cortex involved, in which the vermis and flocculus are relatively spared. The climbing fiber output of the inferior olivary nucleus to the cerebellum is mainly directed to the cerebellar hemispheres. The flocculus and nodulus, both relatively spared in PCH-2 and in OPCH, are reciprocally connected to the vestibular nuclei [45]. We have not found abnormalities in the vestibular nuclei in our series.
In addition to cortical segmental loss, a more diffuse kind of degeneration is found, which includes loss of cortical elements such as Purkinje cells and granule cells. This lends a “moth-eaten” aspect to the myelinated nerve bundles in the cores of folia and central cerebellar white matter, similar to the more common cerebellar degenerations such as in spino-cerebellar ataxias. Some findings such as gliosis in the central parts of the cerebellum and axonal swelling suggest an ongoing process. Study of the cell somata of the nucleus dentatus reveals some peculiarities in all patients where the nucleus could be identified, not seen in controls, such as perisomatic “vacuolation” that was even seen on routine HE staining. The presence of calbindin in some of these “vacuoles” suggests swollen nerve endings.
Genetics and epigenetics
Varying degrees of hypoplastic folial development are found in three patients (4, 5, 6) who share the same ancestors (Fig. 3c–h). This indicates that modifying factors bear on the expression of this genetic condition. Patient 3, the patient with the longest survival, who died at 22 years, differed from other patients in having vascular anomalies in the cerebellum. This phenomenon may be caused by an unknown age-bound complication of the disease. However, a genotype distinct from the others cannot be excluded pending the unraveling of the genetic basis of PCH-2.

pontocerebellar hypoplasia
pons
olivary nucleus
cerebellum


Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification

Aims/hypothesis Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD).
Introduction
Diabetic cardiomyopathy (DCM) is a common complication in type 2 diabetes [1]. Changes in myocardial energy metabolism, due to altered substrate handling, characterise diabetes-related heart disease and DCM [1]. Studies in rodent models, including rats fed a high-fat diet (HFD) [2], leptin-deficient and -resistant animals [3–6] and transgenic mice with cardiac-restricted peroxisome proliferator activated receptor α (PPARα) overexpression [7], indicate that intramyocardial accumulation of triacylglycerol metabolites (lipotoxicity) and myocardial insulin resistance may underlie diabetes-related cardiac dysfunction [1, 8].
Among the putative causes of myocardial triacylglycerol accumulation are elevations in long-chain fatty acid (LCFA) uptake [6, 7, 9]. In the heart, approximately 50% of LCFA uptake is mediated by the fatty acid translocase CD36 [10, 11]. Under physiological conditions, both insulin and contraction stimulate LCFA uptake by inducing the translocation of CD36 from an intracellular pool to the sarcolemma, illustrating that CD36 is also directly involved in the dynamic utilisation of LCFA by cardiomyocytes. This acute translocation of CD36 involves activation of phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (PKB/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades in response to insulin and contraction, respectively [11].
Interestingly, CD36 deletion rescues the cardiac dysfunction, metabolic abnormalities and myocardial triacylglycerol accumulation observed in transgenic mice with cardiac-restricted PPARα overexpression [7], suggesting a critical role for CD36 in the development of diabetes-related heart disease [12]. We hypothesised that long-term alterations in the functional pool of CD36 lead to myocardial accumulation of (toxic) lipid metabolites and insulin resistance and contribute to the development of diabetes-related heart disease. To examine this, we first measured in vivo cardiac dimensions by echocardiography in rats exposed to a high-fat diet (HFD), which impairs myocardial insulin signalling [2, 13]. Subsequently, isolated cardiomyocytes were used to determine changes in substrate uptake and LCFA metabolism under basal conditions and after incubations with insulin and with oligomycin; the latter compound induces a contraction-like elevation of intracellular AMP levels and concomitant activation of AMPK [14]. Finally, we assessed whether the observed alterations could be ascribed to CD36 in hearts from rats on the HFD by analysing the effects of the CD36 inhibitor sulfo-N-succinimidyloleate (SSO) on myocardial LCFA uptake and utilisation in isolated cardiomyocytes, and by studying the expression and subcellular localisation of CD36 in cardiac ventricular tissue.
Materials and methods
Animals The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH Publication No. 85-23, revised 1996) and was approved by the VU University Medical Centre Animal Care Committee. Adult male Wistar WU rats (n = 31; mean body weight 361 ± 24 g) were purchased from Harlan CBP (Horst, the Netherlands), and fed an HFD or an isocaloric low-fat diet (LFD) [2]. At week 8, rats fasted overnight underwent an OGTT, and post-load blood glucose levels were determined 15, 30, 60, 90 and 120 min after ingestion of 2 g glucose/kg body weight [2]. After 10 weeks on the diet, blood was collected for determination of glucose and insulin levels. Rats were then anaesthetised with an intraperitoneal injection of sodium pentobarbital, and hearts were removed for the isolation of cardiomyocytes [15].
Diets Experimental diets were obtained from Hope Farms (Woerden, the Netherlands; HFD, catalogue no. 4148.02; LFD, catalogue no. 4148.01). The LFD consisted of 8% by weight of total fat, 22% by weight of protein and 60% by weight of carbohydrate; the HFD contained 25% by weight of fat, 32% by weight of protein and 25% by weight of carbohydrate, as well as more palmitate (91.12 g/kg) and oleate (100.24 g/kg) compared with the LFD (29.12 and 32.08 g/kg, respectively). In the group fed the HFD, 50.4% of the ingested calories were derived from fat compared with 16.4% in the group fed the LFD.
In vivo cardiac function Non-invasive transthoracic echocardiograms were recorded at heart rates of 350–400 beats per minute in rats anaesthetised with 1.5% isoflurane in a mixture of N2O/O2 (1/2, vol/vol) [16] before and 8 weeks after initiation of the diet by an Aloka echo machine (ProSound SSD-4000) using a 13-MHz linear interfaced array transducer. M- and B-mode images were obtained in the parasternal long- and short-axis views of the left ventricle (LV) at mid-papillary level. At end systole (ES) and end diastole (ED), LV lumen diameter (D), LV ventricular diameter (VD) and posterior (PWT) and interventricular septum (IVSWT) wall thicknesses were determined for three cardiac cycles and averaged. The LV dimensions were used to calculate systolic parameters [17]: fractional shortening , where EDD is end diastolic diameter and ESD is end systolic diameter; end diastolic volume (EDV) = [EDD]3; end systolic volume (ESV) = [ESD]3; and ejection fraction . LV mass (LVM) was determined according to the uncorrected cube assumption: , where 1.05 is the specific gravity of the myocardium [18].
Plasma and tissue determinations Blood glucose was measured from tail bleeds using a glucose analyser (HemoCue, Angelholm, Sweden). Plasma insulin was measured using ELISA (EZRMI-13K; Linco Research, St Charles, MO, USA). Myocardial triacylglycerol content was determined in ventricular lysates from a separate set of animals as described in [2].
Treatment of isolated cardiomyocytes Cardiomyocytes were prepared using a Langendorff perfusion system as described in [15, 19]. Isolated cardiomyocytes were incubated for 30 min at room temperature with 0.5% DMSO or SSO [9]. The CD36-specific inhibitor SSO [20] was used to investigate the involvement of CD36 in basal LCFA uptake. SSO was applied at a concentration of 0.4 mmol/l, which effectively inhibits CD36 [15, 21]. Then, cells were washed and incubation was continued for 15 min at 37°C with continuous shaking. Thereafter, the DMSO-treated cardiomyocytes were kept for another 15 min at 37°C and either kept untreated (basal) or incubated with insulin or oligomycin. To achieve optimal stimulation of glucose and LCFA uptake [22], insulin and oligomycin were used at a concentration of 10 nmol/l and 30 μmol/l, respectively. At this concentration, the ATP-synthase inhibitor oligomycin induces an increase in the intracellular AMP/ATP ratio, thereby activating AMPK-mediated signalling pathways without inhibiting oxygen consumption [14].
Glucose uptake, palmitate uptake and oxidation, and incorporation into intracellular lipid pools Following agonist treatment, [3H]-2-deoxyglucose and [1-14C] palmitate uptake were simultaneously determined during a 3-min incubation [15]. Rates of [1-14C] palmitate oxidation (measured as the production of 14CO2) and esterification (measured as incorporation of the 14C label into intracellular phospholipids [PL], and triacylglycerol) were determined after a 20-min incubation [9].
Western blotting Phosphorylation of proline-rich Akt-substrate 40 (PRAS40-Thr246) and acetyl-coenzyme A carboxylase-Ser79 (ACC-Ser79) was determined in homogenates of cardiomyocytes by western blotting using phospho-specific antibodies [9, 13]. PKB/Akt-Ser473 phosphorylation was determined in ventricular homogenates as described in [2]. Expression levels of CD36 were determined by western blotting using the monoclonal CD36 MO25 antibody [23]. Immunoblots were quantified by densitometric analysis of the films [2].
Cardiac morphology and subcellular localisation of CD36 Cardiac ventricular tissue collected in a previous study [2] was routinely embedded in paraffin (Histowax; Leica Microsystems, Wetzlar, Germany). Sections (5 μm) were cut, and mounted on slides coated with 3-aminopropyltriethoxisilane (Menzal, Darmstadt, Germany). After deparaffinisation and rehydration, the slides were used for determination of cardiomyocyte cross-sectional area or immunohistochemical staining of CD36.Cross-sectional area was determined in randomly chosen fields in haematoxylin/eosin-stained slides using NIH image analysis software (National Institutes of Health, Bethesda, MD, USA) for 20–30 cells per heart and normalised to sarcomere length [24].For CD36 localisation, sections were rinsed extensively with Tris-buffered saline (TBS; 50 mmol/l Tris–Cl, 500 mmol/l NaCl, pH 7.6), and incubated overnight with the monoclonal CD36 MO25 antibody [23] diluted 1:2,000 in TBS containing 0.5% Triton X-100, pH 7.6 at 4°C. After several washes in TBS, sections were incubated for 1.5 h at room temperature with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA), diluted 1:250 in TBS, followed by incubation for 1 h at room temperature with avidin–biotin–peroxidase (1:400; ABC Elite kit; Vector Laboratories). Tissue-bound peroxidase was visualised with the 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogen reaction (7.5 mg DAB, 5 μl 30% H2O2 in 15 ml 50 mmol/l Tris–Cl, pH 7.6) for 10 min at room temperature. Sections were then rinsed with distilled water, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Sections were analysed using a Leica DM-LB light microscope (Leica, Rijswijk, the Netherlands). Digital images were taken at ×200 magnification using a Leica DC500 digital camera, and quantified using Quantity One software (Bio-Rad Laboratories, Veenendaal, the Netherlands).
Statistical analysis Unless indicated otherwise, data are expressed as means±SE for the indicated number of animals (n). Statistical analysis was performed in SPSS for Mac OS X version 11.0.4 (SPSS Inc., Chicago, IL, USA). Differences between the LFD and HFD groups were determined using independent t tests. The paired sample t test was used to define the effect of agonist incubations on cardiomyocyte preparations. p < 0.05 was considered as statistically significant.
Results
HFD induces cardiac contractile dysfunction Feeding rats isocaloric LFD or HFD diets for 8 weeks induced similar weight gains in the two groups (Table 1), but mild glucose intolerance, as determined by an oGTT, in HFD- vs LFD-rats (p < 0.05; Fig. 1a). Figure 1b shows the LV dimensions at the start and 8 weeks after initiation of the diet interventions. Baseline parameters were similar in the two groups (Table 1). After 8 weeks on the diet, LVM, ED-VD, EDD and ED-IVSWT showed comparable increases in LFD and HFD hearts (all p < 0.05), whereas ED-PWT was unaffected by the diet (Table 1). In the ES phase, both ES-PWT and ES-IVSWT were increased in LFD hearts only (both p < 0.005; Table 1). ES-VD was similarly affected by LFD and HFD feeding. ESD was increased in LV from HFD-fed vs LFD-fed rats (p < 0.01; Table 1). FS% was increased in LFD hearts (p < 0.05), and decreased in HFD hearts during the course of the diet intervention (p < 0.02; Table 1). Accordingly, EF% was decreased in HFD compared with LFD hearts (p < 0.05; Table 1). Collectively, these data show that HFD induced cardiac contractile dysfunction in rats.
Table 1In vivo cardiac characteristics of rats before and after 8 weeks on an HFD or a LFD StartLFDHFD(n = 24)(n = 8)(n = 16)Physiological parameters Body weight (g)295 ± 4476 ± 8*463 ± 8* Left ventricular mass (mg)637 ± 15890 ± 18*882 ± 32*LV diastolic parameters Posterior wall thickness (mm)1.63 ± 0.031.80 ± 0.071.71 ± 0.05 Lumen diameter (mm)6.92 ± 0.077.55 ± 0.20*7.68 ± 0.10* Interventricular septum wall thickness (mm)1.37 ± 0.031.67 ± 0.08*1.54 ± 0.04* Ventricular diameter (mm)9.80 ± 0.0610.86 ± 0.13*10.89 ± 0.12*LV systolic parameters Posterior wall thickness (mm)2.93 ± 0.063.45 ± 0.12*3.00 ± 0.08** Lumen diameter (mm)3.41 ± 0.103.32 ± 0.254.07 ± 0.12*,** Interventricular septum wall thickness (mm)2.51 ± 0.052.85 ± 0.09*2.67 ± 0.06 Ventricular diameter (mm)8.81 ± 0.079.82 ± 0.12*9.81 ± 0.10* Fractional shortening (%)50.8 ± 1.356.3 ± 2.4*47.1 ± 1.7*,** Ejection fraction (%)87.5 ± 1.091.1 ± 1.484.9 ± 1.0*,**Data are means±SE*p < 0.05 vs start**p < 0.01 vs LFDFig. 1a Blood glucose levels after an oral glucose load in rats fed the HFD (closed circles; n = 16) and the LFD (open circles; n = 8). Data are means±SE. b Representative echocardiographic M-mode images from at least three cardiac contractile cycles at the start of the diet intervention and 8 weeks after initiation of the LFD and HFD, respectively
Insulin action, but not AMPK signalling, is impaired in HFD cardiomyocytes We used cardiomyocytes to unravel the diet-induced molecular alterations in energy substrate uptake and intracellular signalling pathways. Cardiomyocytes were isolated from a separate group of rats, fed either the LFD or the HFD under identical experimental conditions. At killing, body, heart and liver weights were similar between LFD- and HFD-fed rats, whereas perirenal fat pad weight was 50% higher in HFD- than in LFD-fed rats (p < 0.001; Table 2). Furthermore, HFD feeding increased blood glucose levels (p < 0.02) and lowered plasma insulin levels (p < 0.05).
Table 2Characteristics of rats after 10 weeks on a high- or low-fat diet LFDHFD(n = 14)(n = 17)Body composition Body weight at killing (g)465 ± 5472 ± 10 Heart weight (% body weight)0.445 ± 0.0070.431 ± 0.012 Liver weight (% body weight)3.10 ± 0.073.02 ± 0.08 Perirenal fat pad weight (% body weight)1.63 ± 0.082.43 ± 0.14*Plasma characteristics Blood glucose (mmol/l)5.50 ± 0.085.78 ± 0.08** Plasma insulin (pmol/l)269 ± 18216 ± 16***Data are means±SE*p < 0.001 vs LFD**p < 0.02 vs LFD***p < 0.05 vs LFDBasal 2-deoxyglucose glucose uptake rates were 7.3 ± 0.8 and 11.0 ± 1.6 nmol min−1 [g wet cell mass]-1C in LFD and HFD cells, respectively (p = 0.063; Fig. 2). Insulin stimulated 2-deoxyglucose uptake 3.7-fold in LFD cardiomyocytes and 2.2-fold in HFD cardiomyocytes (both p < 0.001 vs basal, p < 0.02 HFD vs LFD), indicating reduced myocardial insulin responsiveness. Oligomycin had similar stimulatory effects on 2-deoxyglucose uptake in HFD and LFD cardiomyocytes (HFD, 1.6-fold stimulation; LFD, 1.7-fold stimulation; both p < 0.05 vs basal).
Fig. 2Rates of 2-deoxyglucose uptake in cardiomyocytes from rats fed the LFD (open bars) and the HFD (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS) or oligomycin (Oli) before measurement of 2-deoxyglucose uptake. Data are expressed as wet cell mass and are means±SE; n = 8. *p < 0.02, agonist effect; #p < 0.02, diet effectActivation of phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (PKB/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades has been implicated in the stimulation of glucose uptake in response to insulin and oligomycin, respectively. We studied whether changes in the activation of these signalling pathways paralleled the observed alterations in glucose uptake rates in HFD cells. Therefore, phosphorylation of PRAS40-Thr246 and ACC-Ser79 were examined as respective distal determinants for PKB/Akt and AMPK activity [13, 25], as the presence of bovine serum albumin (mol. mass 66 kDa) in the cardiomyocyte homogenates interfered with direct measurement of PKB/Akt (mol. mass 64 kDa) and AMPKα phosphorylation (mol. mass 62 kDa). Basal PRAS40-Thr246 phosphorylation was 1.7-fold higher in HFD than in LFD cells (p < 0.05; Fig. 3a,b). Insulin stimulated PRAS40-Thr246 phosphorylation 12.0-fold in LFD cells (p < 0.001) and 4.0-fold in HFD cells (p < 0.001), thus confirming reduced insulin responsiveness. Oligomycin did not affect PRAS40-Thr246 phosphorylation.
Fig. 3Immunoblots (a, c) and quantification of PRAS40-Thr246 (b) and ACC-Ser79 (d) phosphorylation after incubation of cardiomyocytes from LFD- and HFD-fed rats with DMSO (Basal), insulin (INS) or oligomycin (Oli). Data are means±SE; n = 5. *p < 0.05, agonist effect; #p < 0.05, diet effectACC-Ser79 phosphorylation was measured as a determinant of AMPK activity [25]. Basal levels of Ser79-phosphorylated ACC were similar in LFD and HFD cells (Fig. 3c,d). Insulin had no effect on ACC-Ser79 phosphorylation, whereas oligomycin stimulated ACC-Ser79 phosphorylation in LFD and HFD cells to a similar extent (HFD, 3.0-fold stimulation; LFD, 2.9-fold stimulation; both p < 0.001 vs basal), indicating that AMPK signalling was not affected by high-fat feeding.
HFD feeding results in increased CD36-mediated LCFA uptake Basal rates of LCFA uptake were increased 1.4-fold (p < 0.001) in HFD compared with LFD cells (Fig. 4). The CD36 inhibitor SSO reduced basal LCFA uptake by 44% (p < 0.001) and 23% (p < 0.05) in HFD and LFD cardiomyocytes, respectively. Notably, the absolute rates of LCFA uptake were similar between SSO-treated LFD and HFD cells (Fig. 4), suggesting that the increased basal LCFA uptake rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Insulin stimulated LCFA uptake 1.3-fold in LFD cells (p < 0.02 vs basal) but had no effect in HFD cells (p < 0.02 vs LFD; Fig. 4). Oligomycin increased LCFA uptake in LFD and HFD cardiomyocytes to the same extent (Fig. 4; both p < 0.05 vs basal).
Fig. 4Rates of palmitate uptake (a), oxidation (b) and esterification into intracellular triacylglycerols and phospholipids (c) in cardiomyocytes from LFD-fed rats (open bars) and HFD-fed rats (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS), oligomycin (Oli) or SSO before measurement of palmitate uptake. Data are wet cell mass and are means±SE; n = 8. *p < 0.05, agonist effect; #p < 0.05 diet effectWe next examined the metabolic fate of LCFA following their uptake by HFD and LFD cardiomyocytes. Basal rates of LCFA oxidation were reduced by 22% in HFD relative to LFD cells (Fig. 4b; p < 0.01). SSO further lowered cardiomyocyte LCFA oxidation rates by 66% (p < 0.005) and 31% (p < 0.05) in LFD and HFD cells, respectively, but absolute rates of LCFA oxidation were comparable in SSO-treated LFD and HFD cells.The incorporation rate of LCFA into triacylglycerol was increased 1.4-fold (p < 0.05) in HFD compared with LFD cells under basal conditions (Fig. 4c). SSO reduced triacylglycerol formation in both LFD and HFD cells (both p < 0.05 vs basal). Notably, the absolute triacylglycerol esterification rates in SSO-treated cells did not differ between HFD- and LFD-fed rats. Also, the incorporation of LCFA into phospholipids under basal conditions was increased 1.5-fold in HFD vs LFD cells (p < 0.05; Fig. 4c), and was reduced by SSO in both LFD and HFD cells (both p < 0.05 vs basal). Myocardial triacylglycerol content was 181 ± 50 μg/mg protein in LFD rats and 330 ± 114 μg/mg protein in HFD rats (p < 0.02) [2].
HFD induces cardiomyocyte hypertrophy and alters the subcellular localisation of CD36 The inhibitory effect of SSO suggests that the increased basal LCFA uptake and esterification rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Western blot analysis indicated that the levels of CD36 were similar between HFD and LFD hearts (Fig. 5a). Immunohistochemical staining of cardiac LV tissue indicated cardiomyocyte hypertrophy, as reflected by an increased cross-sectional area (p < 0.05 vs LFD; Table 3), and showed a more abundant presence of CD36 at the sarcolemmal membrane, particularly at the intercalated discs, in HFD hearts compared with LFD hearts (p < 0.002 vs LFD; Fig. 5b,c and Table 3).
Fig. 5Expression and subcellular localisation of CD36. Expression of CD36 in ventricular lysates of HFD and LFD rats. Ponceau S staining confirmed that the filters contained equal amounts of ventricular extracts (a). Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from rats fed an LFD (b, d) or HFD (c, e) for 8 weeks. Rats received an i.p. injection of saline (b, c) or insulin (d, e) 30 min before killing. Photographs are representative of two independent experiments performed on three rats per experimental group. Arrows indicate intercalated discs. Scale bar indicates 25 μmTable 3Cardiac morphology and subcellular localisation of CD36 LFDHFD(n = 6)(n = 8)Morphology Cardiomyocyte cross-sectional area normalised to sarcomere length (μm2)214 ± 3.5276 ± 23*CD36 localisation Sarcolemma  Saline (%)61.7 ± 374.8 ± 1**  Insulin (%)75.9 ± 4***78.2 ± 3 Cytoplasm  Saline (%)38.3 ± 325.2 ± 1**  Insulin (%)24.1 ± 4***21.8 ± 3Data are means±SE*p < 0.05 vs LFD**p < 0.002 vs LFD***p < 0.01 vs salineAs insulin stimulated LCFA uptake in isolated cardiomyocytes (Fig. 4), we also examined the subcellular localisation of CD36 in hearts isolated 30 min following an intraperitoneal injection with insulin (10 U/kg body weight) [2]. Figure 5d shows that in vivo insulin treatment increased CD36 immunoreactivity at the intercalated discs in LFD hearts (p < 0.01 vs saline; Table 3). In HFD hearts, insulin did not further recruit CD36 to the sarcolemma (Fig. 5e).
HFD-induced CD36 redistribution to the sarcolemma precedes the onset of cardiac contractile dysfunction and associates with elevated basal phosphorylation of PKB/Akt We next analysed whether HFD-induced alterations in CD36 localisation precede the onset of cardiac contractile dysfunction, using rats fed an LFD or HFD for 4 weeks. This period was chosen based on serial echocardiographic measurements, which revealed a tendency towards decreased cardiac contractile function 4 weeks after initiation of the diet intervention. Specifically, FS% was 4.2% lower (p = 0.07) and EF% was 3.3% lower (p = 0.10) in HFD vs LFD hearts after 4 weeks on the diet (data not shown), compared with the 9.2 and 6.2% decreases observed after 8 weeks of the feeding regime (both p < 0.05; Table 1). Despite the absence of significant effects on cardiac function after 4 weeks of HFD feeding, immunohistochemical staining of cardiac LV tissue showed that the abundance of CD36 at the sarcolemmal membrane was already markedly increased (Fig. 6a,b) to levels almost similar to those observed after 8 weeks of HFD feeding (Fig. 5b,c).
Fig. 6Effect of the duration of HFD feeding on CD36 localisation and phosphorylation of PKB/Akt. Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from saline-injected rats fed an LFD (a) or HFD (b) for 4 weeks. Photographs are representative of two or three independent experiments performed on six rats per experimental group. The arrows indicate intercalated discs. The scale bar indicates 25 μm. c Immunoblot and quantification d of PKB/Akt-Ser473 phosphorylation levels in ventricular homogenates from saline-injected rats fed an LFD (open bars) or HFD (filled bars) for 4 and 8 weeks, respectively. Data are means±SE, n = 8. *p < 0.05, diet effectFinally, we analysed whether HFD feeding induced changes in the activity of signalling pathways implicated in CD36 trafficking. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PKB/Akt, respectively [26]. As insulin does not further recruit CD36 to the sarcolemma in HFD hearts (Fig. 5e), we proposed that increases in basal activity of PKB/Akt may underlie the redistribution of CD36 in HFD hearts. Phosphorylation of PKB/Akt was determined in LV homogenates of the same group of saline-injected rats in which CD36 localisation was measured. As shown in Fig. 6c,d, basal phosphorylation of PKB/Akt was 1.7- and 2.6-fold higher after 4 and 8 weeks of HFD feeding, respectively (both p < 0.05). Collectively, these results indicate that increased activity of PKB/Akt may underlie CD36 redistribution to the sarcolemma, and that these events precede the LV dysfunction induced by HFD feeding.
Discussion
Here we report that HFD feeding induces cardiac contractile dysfunction in rats and that this is associated with a permanent relocation of CD36 to the sarcolemma. The continuous presence of CD36 at the sarcolemmal membrane results in enhanced rates of LCFA uptake and subsequent esterification. We propose that this contributes to a decrease in myocardial insulin action and the development of diabetes-related heart disease. In addition, we show that AMPK-mediated responses are not affected by the composition of the diet.
A key observation in this study is that the alterations in cardiac contractile function in HFD hearts was associated with a continuous presence of CD36 at the sarcolemmal membrane. The present study provides the first morphological evidence for translocation of CD36 to the sarcolemmal membrane. Importantly, the amount of sarcolemmal CD36 closely correlated with enhanced LCFA uptake rates in isolated cardiomyocytes. The observation that SSO inhibited LCFA uptake in HFD cardiomyocytes to the same residual levels as measured in LFD cardiomyocytes provides further pharmacological evidence that the enhanced flux of LCFA in the heart of HFD-fed rats is a direct consequence of the relocalisation of CD36 to the sarcolemmal membrane.
Previously, a redistribution of CD36 to both subsarcolemmal and intramyofibrillar mitochondria has also been observed [27, 28]. Can the observed CD36 immunoreactivity be ascribed to subsarcolemmal mitochondria rather that the sarcolemma itself? Stimuli inducing translocation of CD36 to the mitochondria are expected not to discriminate between the subsarcolemmal and the intramyofibrillar mitochondria. Hence, if a stimulus or condition, in this case insulin or HFD feeding, were to increase the subsarcolemmal CD36 content, one would expect a similar increase in intramyofibrillar CD36 content. This, however, could not be confirmed in the immunohistochemical experiments. Furthermore, biochemical fractionations performed in previous studies substantiate the idea that a significant fraction of CD36 is present at the sarcolemma of hearts from insulin-resistant rats, and that only a minor portion of CD36 is found within the mitochondrial fractions [9, 22]. While insulin has been shown to translocate CD36 to the sarcolemma [9, 22], insulin-mediated CD36 translocation to the mitochondria has never been reported and even seems counterintuitive. Collectively, the available evidence strongly supports the idea that the observed CD36 immunoreactivity can be ascribed to CD36 located at the sarcolemma rather than in subsarcolemmal mitochondria.
The combined data of this and our earlier study on obese Zucker rats [9] suggest that relocalisation of CD36 is a general phenomenon in insulin-resistant hearts, and raises the question of what mechanism underlies the continuous presence of CD36 at the sarcolemmal membrane in HFD hearts. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PI3K/PKB/Akt, respectively [26]. Activation of AMPK is critical for contraction- and oligomycin-mediated CD36 translocation, whereas PI3K/PKB/Akt is critical for insulin-mediated CD36 trafficking [10, 14, 26]. Basal phosphorylation of PKB/Akt and its distal target, PRAS40, were elevated in HFD compared with LFD hearts, whereas AMPK activity was not affected by the diet. Furthermore, in cells from HFD-fed rats, the stimulatory effects of insulin on LCFA uptake, CD36 translocation and phosphorylation of PKB/Akt and PRAS40 were abrogated, whereas oligomycin-induced AMPK activation and LCFA uptake was unimpaired between cardiomyocytes from LFD- and HFD-fed rats. Although we cannot exclude a contribution of other kinases or HFD-induced alterations in the as yet undisclosed trafficking machinery regulating the internalisation of CD36 [11, 26], our observations do not argue against the suggestion that increases in PKB/Akt activity may contribute to the sustained sarcolemmal presence of CD36 in the heart of HFD-fed rats.
Previously, we suggested that increased plasma insulin might contribute to CD36 relocalisation in hearts from obese Zucker rats [9]. As systemic hyperinsulinaemia was not observed in HFD-fed rats [2, 13], it remains interesting to examine whether changes in the activity of other (insulin-independent) regulators of PKB/Akt phosphorylation, such as PI3Kγ- and β2-adrenergic receptor signalling pathways, Ca2+-calmodulin dependent kinase, protein phosphatase 2A and the sympathetic nervous system [29], can be linked to the observed increase in PKB/Akt phosphorylation in HFD hearts.
The metabolic and biochemical data were paralleled by in vivo cardiac functional changes. Importantly, HFD-induced cardiac contractile dysfunction does not seem to be linked to diet-related elevations in blood pressure. Circadian haemodynamic parameters were monitored using implanted telemetry devices in an experiment performed in a separate group of animals. We found a slight increase in night-time (activity-related) heart rate in HFD rats, but no changes in blood pressure even after 10 weeks of exposure to the diet (Electronic supplementary material Table 1). Others confirmed the absence of relevant hypertension after feeding rats a diet containing 74% fat for 17 weeks [30]. This underscores the possibility that alterations in metabolism, rather than in haemodynamics, underlie the impairment in cardiac contractile function in HFD rats.
The present study also suggests that HFD-induced alterations in the functional pool of CD36 may contribute to an imbalance in LCFA uptake and in oxidation and esterification rates in HFD vs LFD cells. Whereas LCFA uptake rates were increased 1.4-fold in HFD cells, the LCFA oxidation rates were not increased but rather modestly lower in HFD cells, while the rates of esterification into triacylglycerol and phospholipids were 1.4-fold higher in HFD vs LFD cardiomyocytes. Accordingly, triacylglycerol content was 1.9-fold increased in HFD compared with LFD hearts [2]. It seems unlikely that changes in malonylCoA levels underlie the reduced rates of LCFA oxidation in HFD hearts, as no diet-induced changes in ACC phosphorylation were found. Rather, degenerative changes in mitochondria, such as matrix dilution, cristolysis and mitochondria-associated lamellar bodies, have been observed in cardiomyocytes from HFD-fed rats [2] and have been linked to reduced oxidative capacity and lipid accumulation in skeletal muscle from patients with type 2 diabetes. However, further studies are required to examine whether these changes contribute to the reduced basal LCFA oxidation and increased LCFA esterification rates, or whether the extra LCFA taken up by HFD cells are stored as triacylglycerol and phospholipids as a consequence of HFD-induced changes in enzymes promoting LCFA esterification or inhibition of lipolysis.
An important finding of this study is that the HFD-induced CD36 redistribution preceded the onset of cardiac contractile dysfunction. Although we cannot unequivocally link CD36 redistribution to cardiac dysfunction, a recent report provided strong support for this notion as the absence of CD36 was found to prevent myocardial triacylglycerol accumulation in transgenic mice with cardiac PPARα overexpression, both under normal conditions and after HFD feeding [12]. Similarly, alterations in PKB/Akt activity have been linked to cardiac dysfunction [31]. Myocardial biopsy samples obtained from patients with advanced heart failure or dilated cardiomyopathy show increased basal phosphorylation of PKB/Akt [32, 33], and transgenic mouse models with constitutively activated PKB/Akt in the heart develop hypertrophy, decreased cardiac function and impaired recovery from ischaemia–reperfusion injury [31, 34]. Whereas chronic activation of PKB/Akt increases basal glucose uptake rates in the heart [35], the effects on myocardial lipid metabolism have not been studied. Interestingly, PKB/Akt signalling has been linked to palmitate-induced beta cell lipotoxicity [36]. Based on this report and the findings described in this study, it would be of interest to analyse animals expressing chronically active PKB/Akt for translocation of CD36 to the sarcolemma and myocardial lipid accumulation.
It seems plausible that an enhanced CD36-mediated LCFA uptake rate may contribute to the development of cardiac contractile dysfunction. An increased supply of LCFA may influence excitation–contraction coupling as well as other processes linked to cellular Ca2+ handling [37, 38]. Furthermore, triacylglycerol accumulation is strongly linked to cardiac insulin resistance and contractile dysfunction [1, 2, 8]. The dynamic equilibrium between triacylglycerol stores and triacylglycerol metabolites causes accumulation of ceramide and diacylglycerol during prolonged LCFA influx. Both ceramide and diacylglycerol have been implicated in the activation of serine/threonine kinases, such as PKC, Jun N terminal kinase (JNK), and inhibitor of nuclear factor-κB kinase (IKK), which counteract insulin signalling [39, 40]. Notably, chronic myocardial activation of Akt in transgenic mice has also been linked to feedback inhibition of PI3K activity [33] and blunted insulin stimulation of glucose uptake [35]. Importantly, reduced insulin responsiveness reduces the ability of insulin to regulate substrate handling, e.g. metabolic inflexibility [41]. In this study and previous reports [2, 13], we show that HFD feeding reduces insulin responsiveness and impairs metabolic flexibility, as illustrated by the blunted effects of insulin on the stimulation of glucose uptake, phosphorylation of PRAS40, LCFA uptake and translocation of CD36 to the sarcolemma.
We conclude that HFD feeding in rats induces cardiac contractile dysfunction, which is preceded by relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The continuous presence of CD36 at the sarcolemma contributes to enhanced rates of fatty acid uptake, resulting in myocardial triacylglycerol accumulation and accompanying insulin resistance. Collectively, these data suggest that alterations in the subcellular localisation of CD36 may contribute to the development of diabetes-related heart disease and that CD36 may be a therapeutic target to prevent cardiac dysfunction and the development of heart failure in diabetes.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM Table 1
Heart rate and blood pressure of rats after 10 weeks on an HFD or a LFD (PDF 22 kb)

contractile dysfunction
cd36
diabetic cardiomyopathy
insulin resistance
glucose uptake
high-fat feeding
fatty acid utilisation


Complications and reoperations of the SB Charité lumbar disc prosthesis: experience in 75 patients

Artificial disc prosthesis show fair to good short- and mid-term results. Long-term results are becoming apparent now, however, the incidence of late complications with this procedure remain poorly understood. In this report we will analyse late complications and discuss our experiences with salvage operations in patients with persistent pain after SB Charité disc prosthesis implantation. Seventy-five patients with persistent leg and back pain after insertion of an artificial disc prosthesis were enrolled in the study. In this negative selection frequently occurring late-complications were subsidence, wear, adjacent disc degeneration, facet joint degeneration and migration. In 15 patients we performed a posterior fusion without disc removal, and in 22 patients we removed 26 prostheses and performed a posterior and anterior fusion. The visual analogue scale (VAS) and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year, which is not yet available in all patients. The VAS and Oswestry decreased in the posterior group (n = 10) respectively from 8.0 (SD 0.9) to 6.3 (SD 2.1) and from 57.0 (SD 17.0) to 44.6 (SD 20.4); and in the disc removal group (n = 14) respectively from 8.0 (SD 0.9) to 5.6 (SD 2.7) and from 56.3 (SD 14.0) to 43.0 (SD 20.7). Serious late complications may occur following total disc replacement. Removal of the SB Charité artificial disc is feasible but with inherent risks. Removal of the disc prosthesis gives slightly better results than posterior fusion alone after a follow-up of at least 1 year.
Introduction
Degenerative disc disease (DDD) is a major cause of pain and disability, with great social and financial impact, playing an increasing role in modern society [21]. Several surgical techniques have been developed to treat DDD. Spinal fusion is seen as the “gold standard”, but nowadays artificial disc prostheses are an alternative method [13]. The artificial disc should preserve motion, stability and normal function of a spinal segment. Also, less adjacent segment degeneration is expected [6, 7, 13, 15]. In theory, this procedure has many advantages over spinal fusion. A spinal fusion does eliminate motion and can cause overloading and early degeneration of the adjacent levels, although usually appearing only after 10–15 years [1, 11, 12, 17].
Results after implantation of the SB Charité artificial disc prosthesis are diverse. short- and mid-term results are fair to good [3, 5, 13, 22]. The FDA-IDE study, in which the SB Charité artificial disc was tested for approval in the US, showed a FDA-defined, overall clinical success rate of 57.1% after 2-year follow-up [2, 15]. For the IDE study, the procedure was only judged to be successful if four criteria were satisfied: (1) greater than 25% improvement in Oswestry disability index; (2) no device failure; (3) no major complications; and (4) no neurological deterioration compared to preoperative status. Consequently, the composite definition of success employed in the IDE study makes it difficult to compare with other studies that employed different success criteria.
In a 10-year follow-up period, Lemaire et al. [13] found that 90% of patients had good to excellent clinical outcome. On the other hand, in the only available long-term study of Putzier et al. [16] with an average follow-up of 17 years, the investigators found no evidence that long-term results of the disc prosthesis were superior to spinal fusion [16]. It remains unclear whether the early fair to good results obtained with an artificial disc will be consistently maintained with a longer follow-up period [9, 16, 18, 20].
In the Netherlands, more than 1,000 patients have been implanted with a SB III Charité (Link, Germany) disc prosthesis starting in 1989. As these patients received the implant during routine clinical practice, they were not the subjects of a randomized trial. However, 50 patients (75 arthroplasties) were enrolled in a prospective observational trial, and at 2 years the clinical success rate was found to be 70% [22].
Since 1989, there have been many changes in the available implant sizes, surgical instrumentation, and patient indications. Although the basic “SB III” design has remained the same throughout the past 18 years, there have also been evolutionary changes in the polyethylene (PE) resin, sterilization, and endplate fixation technology. Starting in 2004, the SB III design has been renamed the Charité artificial disc and is currently produced by a different manufacturer (DePuy Spine, Raynham, MA, USA). Despite these aforementioned changes, it remains crucially important to fully understand the long-term clinical failure modes of early total disc replacements, as they form the foundation for judging the performance of contemporary implant designs and treatment paradigms.
At present, we have treated 75 patients at our clinic with persistent leg and back pain after insertion of the SB Charité disc prosthesis. The incidence of complications following Charité artificial disc implantation at our institution has proven difficult to deduce because all operations were performed elsewhere. At 2 years of follow up, investigators from the hospital implanting the disc prosthesis reported that 17 out of 50 (34%) patients required secondary surgery, and there were three (6%) reported serious complications [22]. The purpose of this study is to analyse late-complications after insertion of a disc prosthesis, and to describe our experiences with salvage operations in this difficult patient group.
Materials and methods
Patients
Approximately more than 1,000 Dutch patients, suffering from serious and constant back and leg pain, have been implanted with a SB III Charité (Link, Germany) artificial disc prosthesis in the lower lumbar spine.
Over the last 10 years, 75 patients of this cohort, with persisting back and leg pain and being unsatisfied with their clinical situation, were seen at the orthopaedic outpatient clinic of the University Hospital of Maastricht (UHM). They were seen by a different orthopaedic surgeon (AvO and LvR) than the surgeon who performed the prosthesis implantation. Twenty-seven of these cases have already been reported previously [20].
Forty-one patients were female with an average age at the time of disc implant of 42 years and 7 months (30–63 years) and 34 patients were male with an average age of 40 years and 9 months (30–51 years) at the time of disc implant (Table 1). The operated levels and the year the patients received their artificial disc implantation is shown in Table 1.
Table 1Patient characteristicsSexNAge at artificial disc insertionMales3440 years and 9 months (30–51)Females4142 years and 7 months (30–63)Operated level  First level  L2–L31  L3–L43  L4–L522  L5–S130 Second levels   L3–L4, L5–S11  L4–L5, L5–S116Third levels   L3–L4, L4–L5, L5–S11  L2–L3, L4–L5, L5–S11Time disc implantation 1989–199425 1995–199930 2000–200520
Forty-six out of these 75 patients needed one or more salvage operations after their artificial disc implant. Indicators for reoperations were primary absence of pain relief, new pathology according to radiography, CT-scan or MRI in adjacent segment(s), subsidence, facet joint arthrosis or migration of the prosthesis. In our hospital, we performed posterior fusion in 15 patients without disc removal, and in 22 patients we removed 26 prostheses and performed an anterior and posterior fusion. In addition, seven patients received posterior fusion elsewhere, and in two patients the disc prosthesis was removed elsewhere (Fig. 1).
Fig. 1Overview of reoperations after disc prosthesis implantation (UHM: University Hospital Maastricht)
Surgical method of disc prosthesis removal
The disc prosthesis was removed by a team consisting of a vascular surgeon and an orthopaedic surgeon. The patient was placed under general anaesthesia and positioned in supine position (for level L5–S1) or in a semilateral position (for level L4–5 or higher).
A disc prosthesis at level L4–5 was removed via anterolateral lumbotomy, without mobilisation of the great vessels. A disc prosthesis at level L5–S1 was removed through the original left retroperitoneal approach or alternatively via right retroperitoneal, between the great vessels. To remove the disc prosthesis one of the endplates was released by undercutting and removal of some bone after cleaning of fibrous tissue. The metal endplate was gripped and removed with twisting and pulling manoeuvres. Then the core and second endplate was easily released and removed.
The gap between the vertebral bodies after retrieval of the disc prosthesis was mostly 2.5–3 cm high. This was filled with an autologous strut graft in 2 patients, artificial bone in 3 patients and allograft strut grafts and allograft bone chips from femoral heads in 17 patients. We now favour the insertion of allograft strut grafts from the bone bank.
Clinical performance and complications
Before the orthopaedic surgeons in our outpatients clinic performed reoperation, they evaluated the images of X-ray, CT-scans, MRI and if necessary discography on the presence of late complications.
Clinical examination included the ten-point visual analogue scale (VAS), ranging from 0 points for “no pain” and 10 points for “severe pain”, and the Oswestry low back pain disability questionnaire. The VAS and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year after this operation. The 1 year follow-up VAS and Oswestry are not yet available in all patients.
Statistical analysis
Analyses were performed using SPSS, release 12.0.1. Non-parametric tests, i.e. Mann–Whitney and Wilcoxon, were used to test mean. Significance was indicated by P values less than 0.05.
Results
Late complications
An overview of late complications after disc implantation is shown in Table 2. They included the following: 39 patients showed subsidence of the disc prosthesis, in 24 of these patients the disc prosthesis was considered too small. Thirty-six patients showed signs of adjacent disc degeneration, narrowing of the disc and osteophytes on conventional X-rays. In 17 patients this was not obvious before artificial disc insertion on plain X-rays and discography. Eleven patients with multi-level adjacent disc degeneration had developed degenerative lumbar scoliosis. In 25 patients facet joint degeneration was seen on CT-scans. In six patients the disc prosthesis showed anterior migration and in two patients posterior migration of the disc prosthesis occurred. In ten patients we discovered breakage of the metal wire around the core (Fig. 2).
Table 2Overview of late complications after receiving a disc prosthesis (patients can have more than one complication)Late complicationsNumber of patientsSubsidenceDisc prosthesis too small3924Adjacent disc degeneration36Degenerative scoliosis11Facet joint degeneration on CT scan25Anterior migration6Posterior migration2Breakage metal wire10Wear5Severe osteolysis1Subluxation PE core1Fig. 2Example of removed polyethylene core L4–5 (left) and L5–S1 (right) (6.5 years after insertion) with a fracture of the metal wire and damage of disc prosthesis
Less often the surgeons noticed the subsequent late complications. In one case severe osteolysis was seen in the sacrum in a ruptured and severely worn L5–S1 case. In another case a subluxation of the PE core and an adjacent osteoporotic compression fracture was noticed. In five patients radiological wear of the disc prosthesis was obvious due to loss of height of the core, or sclerosis and cysts around the prosthesis on CT-scan.
Study population
The group of 15 patients receiving posterior fusions in our hospital without removing the prosthesis, consisted of 8 men and 7 women. Mean age at their revision surgery was 49 years and 9 months (34–76 years) and mean time-interval between their disc implant and revision surgery was 7 years and 11 months (2–15 years).
Facet joint degeneration was noticed during all operations. Afterwards, we removed the disc prosthesis in four patients of this group because of persisting pain. Nowadays, we advise disc prosthesis removal in conjunction with fusion surgery, assuming that the disc prosthesis can remain a pain source even after solid posterior fusion.
So far, we retrieved 26 prostheses in 22 patients (17 females, 5 males). The additional posterior fusion took place in nine patients 2 weeks before or 2 weeks after the removal of the disc prosthesis. In the other 13 patients, posterior fusion was done as a second operation on the same day as the removal. Nowadays this is the standard procedure in our hospital. The mean interval between insertion and retrieval of the disc prosthesis was 8 years and 11 months (3–16 years). The mean age at retrieval of the disc prosthesis was 50 years (40–72 years).
A pre- and post-operative radiograph of one case, in which we removed the disc prosthesis L4–5 and L5–S1 and performed a posterior and anterior fusion, is shown in Figs. 3 and 4.
Fig. 3a Anteroposterior and b lateral radiograph of the lumbar spine 3 years and 9 months after disc replacement at L4–5 and L5–S1Fig. 4a Anteroposterior and b lateral radiograph of the lumbar spine 9 months after revison surgery
Intraoperatively we twice encountered a lesion of the left common iliac vein (in L5–S1 cases), once a lesion of the left common iliac artery (in a L4–L5 case) and once a small incomplete colon lesion. These complications could all be controlled by the vascular surgeon with relatively little blood loss. Mean blood loss for the anterior procedure was 753 cc (60–5,100 cc). In one patient profound bleeding was encountered from the vertebral body bone and possibly the epidural plexus underneath the distal endplate of a L5–S1 disc prosthesis. This was controlled by packing with bone bank chips and gel foam, however the total blood loss in this two-level case was 5,100 cc.
In another patient we planned to remove the disc prosthesis, however, due to a rupture of the small intestine during the access phase, we decided only to perform a posterior fusion.
Preliminary clinical results
General clinical information for both revision strategies, with a follow-up period of at least 1 year, is shown in Table 3.
Table 3Clinical information for both revision strategies (with a follow-up period of at least 1 year)NrSexYear index surgeryAge index surgeryLevelsYear re-operationRemoval/fusionExtra procedures% improvement VAS% improvement OswestryClinically improved Oswestry (>25%)1F199344L2–L32003FusionNone41.1827.50Yes2M199541L4–L51997FusionNone−33.33−44.44No3F199263L5–S12005FusionNone−14.29−24.34No4M199730L5–S12001FusionNone27.7816.67No5F200042L4–L52003FusionNone12.5014.71No6F199642L4–L5, L5–S12003FusionNone27.786.45No7F200238L4–L5, L5–S12003FusionNone29.4116.13No8M199748L5–S12004FusionNone86.6782.76Yes9M199537L5–S12005FusionNone18.750.00No10F199734L5–S12002Fusion None0.0074.07Yes2005RemovalNone29.41MissingMissing11M199237L4–L52005RemovalNone−12.5025.00Yes12F199533L4–L52006Removal None−12.505.41No13M198944L4–L52005RemovalNone12.5030.00Yes14F199139L4–L52002RemovalNone87.5056.25Yes15F199539L5–S12004RemovalNone76.4773.08Yes16F199946L4–L5, L5–S12005RemovalNone62.5062.50Yes17F199233L3–L4, L5–S12005RemovalNone11.1112.50No18M199846L4–L52004RemovalNone9.0938.10Yes19F199232L2–L3, L4–L5, L5–-S12004RemovalNone55.5616.80No20M200147L4–L52005RemovalNone−6.670.00No21M199539L4–L52005RemovalDynesys62.503.33No22F200237L4–L5, L5–S12005RemovalNone11.1110.00No23F199055L4–L52006RemovalNone14.2913.97No
Ten patients receiving posterior fusion without removal of the prosthesis, have at this moment a follow-up period of more than 1 year since their posterior fusion. The mean VAS before posterior fusion was 8.0 (SD 0.9) and after posterior fusion 6.3 (SD 2.1) (Fig. 5).
Fig. 5VAS scores before and after salvage operation
Furthermore, 14 patients had a follow-up period of more than 1 year since their disc prosthesis removal. Two disc removal patients had insertion of the Dynesys fixation system (one patient had a follow-up of at least 1 year), in addition to the fused disc prosthesis level, for multiple adjacent levels degeneration. This multilevel Dynesys instrumentation was recently removed in both patients due to screw loosening. The VAS score in this group decreased significantly from 8.0 (SD 0.9) before disc prosthesis removal to 5.6 (SD 2.7) after removal (P < 0.05) (Fig. 5). The percentage of improvement after revision surgery in both groups is shown in Fig. 6.
Fig. 6Percentage change in VAS scores in both revision strategies
The mean Oswestry decreased in the posterior fusion group (n = 10) from 57.0 (SD 17.0) to 44.6 (SD 20.4), and in the disc removal group (n = 13) from 56.3 (SD 14.0) to 43.0 (SD 20.7) (Fig. 7). This questionnaire is missing in one patient from the disc removal group. According to the abovementioned IDE-criteria, in which an improvement of ≥25% was considered to be clinically improved, 3 out of 10 patients in the fusion group and 6 out of 13 patients in the disc removal group were clinically improved (Table 3; Fig. 8).
Fig. 7Oswestry scores before and after salvage operationFig. 8Percentage change in Oswestry scores in both revision strategies
Postoperative complications
Two patients from the posterior fusion group developed pseudo-arthrosis postoperatively.
We encountered five postoperative complications in the disc removal group. One patient developed deep venous thrombosis (DVT) of the left leg after suturing a left common iliac vein lesion. In two patients, decreased sensitivity in the left groin and upper leg was noticed, which was partially reversible. Two patients have severe pain and decreased diffuse strength in the left leg postoperatively. In one of these patients these complaints are diminishing at the moment. Presumably, excessive retraction of the lumbosacral nerves in the psoas muscle played a role in these left leg complications.
Discussion
Seventy-five patients with persistent leg and back pain after insertion of SB Charité disc prosthesis were studied. The causes of persisting pain were thought to be related to the following late-complications: subsidence, migration, wear of the disc prosthesis, facet joint degeneration or adjacent degeneration in various combinations.
Good placement and good sizing of the disc prosthesis appeared problematic for many of the patients in our series. In 39 patients we saw subsidence of the disc prosthesis, and in our judgement X-rays showed that in 24 of these patients the disc prosthesis was too small. Whether suboptimal sizing and placement resulted from initial surgical technique, or from historical limitations in instrumentation and/or sizing availability remains unclear. Fixation of the disc prosthesis can be inadequate, giving subsidence and migration of the disc prosthesis. Migration can probably be prevented by coating of the metal plate, but subsidence probably not.
Previous studies suggested that adjacent degeneration is prevented after disc replacing surgery [4, 8, 14]. However, in our study 36 patients showed adjacent disc degeneration. This could be the result of the DDD itself, spreading to multiple levels of the spine, and/or be the consequence of stresses on adjacent levels, generated from the unphysiological motion and functioning of the disc prosthesis.
Concerning the 25 patients in which facet joint degeneration was visible on CT-scans, we hypothesized that an instability is created after removal of the anterior longitudinal ligament and the annulus fibrosis. Replacement with an unconstrained prosthesis will accentuate this instability, especially in extension and axial rotation. The facet joints will be the only restrictor of axial rotation and will degenerate with time.
Because of persisting back and leg pain we performed a posterior fusion without disc removal in 15 patients. Initially, the results were disappointing in most patients, and we therefore started to remove the disc prosthesis. In all patients with removal of the disc prosthesis, PE wear was observed in minor or major degree with surrounding inflammatory fibrous tissue containing PE wear debris. The association between periprosthetic tissue inflammatory reactions and clinical manifestations of pain is not clear at the present time and will be studied further. It is speculated that with removal of the disc prosthesis and the inflammatory periprosthetic tissue, the patient may obtain better pain relief than after only posterior fusion [19]. This hypothesis obviously needs further support with a larger number of more carefully selected patients.
Clinical results after disc prosthesis revision performed in our hospital were diverse. Patients with short segment fusions seemed to fare better than patients with long segment fusions or long flexible fixations with the Dynesys system. At present, the small number of patients and the large number of potential factors influencing the outcomes (Table 3), precluded assessment of a significant association between revision strategy and outcome in the current study.
The mean VAS for pain after disc prosthesis removal was reduced significantly with 2.4 points, from 8.0 to 5.6, whereas the VAS in patients with posterior fusion showed a smaller decrease from 8.0 to 6.3. However, the analyses are based on a small number of cases and a greater number of patients with longer follow up are necessary for a more definitive conclusion.
In a collaborative study by Kurtz et al. [10] it was concluded that artificial discs exhibited wear mechanisms similar to artificial hips and knees. Since the operated patients are mostly between 30 and 50 years at the time of their disc implantation and these young patients will be likely very active, wear may become a clinically significant issue at long-term follow-up. The clinical relevance of wear in total disc replacements is the subject of intense, ongoing research at our institutions.
A major point to consider is that, in contrast with a posterior fusion, retrieval of an artificial disc prosthesis can be dangerous because of nearby vascular structures and scar tissue from the original surgery. However, in our hands, disc removal surgery proved feasible in all but one case in this patient group (26/27 disc retrievals, 96%). Due to the availability of a vascular surgeon during the retrieval surgery average blood loss could be restricted.
In conclusion, serious complications may arise following total disc replacement surgery, however, as yet relatively few data are available to provide the basis for treatment of patients with clinically failed artificial discs. Removal of the disc prosthesis with anterior and posterior fusion gives slightly better results than posterior fusion alone after a follow-up of at least 1 year. Removal of the SB Charité artificial disc was feasible but with inherent risks. As more data become available, perhaps revision artificial disc surgery may become a more common operation for spine surgeons in the near future.

complications
artificial disc
retrieval surgery
risk of reoperation