Showing papers by "James T. Rutka published in 2009"

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

Abstract: We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

The miR-17/92 polycistron is up-regulated in Sonic hedgehog-driven medulloblastomas and induced by N-myc in Sonic hedgehog-treated cerebellar neural precursors

Abstract: Medulloblastoma is the most common malignant pediatric brain tumour and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution SNP genotyping arrays and subsequent interphase FISH on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated microRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the Sonic Hedgehog (Shh) signaling pathway as compared to other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation, and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.

Natural history and outcome of optic pathway gliomas in children.

Abstract: Background The optimal management of optic pathway gliomas (OPGs) is complicated by their variable natural history, the association with neurofibromatosis type 1 (NF1) and difficulties in defining progression and response to treatment. Methods This study is a retrospective review of all children presenting to a single institution with an OPG between 1990 and 2004. Results Of the 133 children included, 78 (59%) had NF1; 87 (71 NF1) were observed initially, of whom 23 (11 NF1) subsequently required treatment. Forty-six patients received immediate treatment. Initial treatment, without or with an observation period, comprised chemotherapy alone (32, 11 NF1); debulking + chemotherapy (15, 4 NF1); gross total resection (6); radiotherapy (2); debulking + radiotherapy (3); and debulking only (12, 3 NF1). Overall, 16 patients were irradiated during the study period. Four children died (overall survival at 5 and 10 years was 97.6% and 94.6% for those who required treatment). Progression-free survival (PFS) for the 69 patients who needed treatment was 48%. There was no difference in PFS between chemotherapy versus chemotherapy + debulking or debulking alone. PFS for the NF1 patients who required treatment was similar to that of non-NF1 patients. Mean follow-up time was 9.0 (range 0.6–18.0, median 8.6) years. Conclusions The study confirms the complexity of OPGs and that NF1 is a major determinant of the resultant behavior of the tumor. Pediatr Blood Cancer 2009; 53:1231–1237. © 2009 Wiley-Liss, Inc.

The changing epidemiology of paediatric brain tumours: a review from the Hospital for Sick Children.

Abstract: This study examines the changing epidemiology of paediatric brain tumours over the past three decades (1980–2008) in a single institution, SickKids, Toronto, Canada. We classified 1,866 surgical pathology cases of brain tumours in children under the age of 19 according to the World Health Organization 2007 consensus and analysed them by gender, histological tumour type, age distribution and decade. Males showed a slightly higher predominance with 56.8% of cases overall. The main histological tumour types were low-grade (I/II) astrocytomas (26.4%), medulloblastoma (10.6%), anaplastic astrocytoma/glioblastoma multiforme (7.1%) and ependymoma (7.0%). Over three decades, an increasing proportion of certain tumour types, including pilocytic astroctoma, atypical teratoma/rhabdoid tumours and neuronal/mixed neuronal-glial tumours was seen. Our results are consistent with those published with similar methodologies in other countries. Any changes in the epidemiology of childhood central nervous system tumours over the past three decades may be attributed in part to changing classification systems, improved imaging technologies and developments in epilepsy surgery; however, continued surveillance remains important.

Neurosurgical management of intractable rolandic epilepsy in children: role of resection in eloquent cortex. Clinical article.

Abstract: Object The authors undertook this study to review their experience with cortical resections in the rolandic region in children with intractable epilepsy. Methods The authors retrospectively reviewed the medical records obtained in 22 children with intractable epilepsy arising from the rolandic region. All patients underwent preoperative electroencephalography (EEG), MR imaging, prolonged video-EEG recordings, functional MR imaging, magnetoencephalography, and in some instances PET/SPECT studies. In 21 patients invasive subdural grid and depth electrode monitoring was performed. Resection of the epileptogenic zones in the rolandic region was undertaken in all cases. Seizure outcome was graded according to the Engel classification. Functional outcome was determined using validated outcome scores. Results There were 10 girls and 12 boys, whose mean age at seizure onset was 3.2 years. The mean age at surgery was 10 years. Seizure duration prior to surgery was a mean of 7.4 years. Nine patients had preoperativ...

Neurosurgical implications of achondroplasia.

Abstract: Object Achondroplasia is the most common form of human short-limbed dwarfism. The pediatric neurosurgeon is frequently required to treat children with achondroplasia who have hydrocephalus, cervicomedullary compression (CMD), and spinal canal stenosis. Accordingly, the authors have reviewed the experience of neurosurgery in children with achondroplasia at The Hospital for Sick Children. Methods The medical records and neurosurgery database at The Hospital for Sick Children were searched to identify all children with achondroplasia who underwent at least 1 neurosurgical procedure between 1956 and the present. Results Twenty-nine children with achondroplasia underwent 85 surgical procedures: 52 for CSF diversion in 12 patients, 20 for CMD in 18 patients, 8 for spinal disorders in 4 patients, and 5 for miscellaneous purposes in 4 patients. The CSF shunts were placed almost exclusively before 1990 and were associated with a significant number of complications. Patients undergoing CMD did very well, with only ...

Epilepsy surgery in the first 3 years of life: A Canadian survey

Abstract: Summary Objective: To determine the clinical characteristics, surgical challenges, and outcome in children younger than 3 years of age undergoing epilepsy surgery in Canada. Methods: Retrospective data on patients younger than age 3 years who underwent epilepsy surgery at multiple centers across Canada from January 1987 to September 2005 were collected and analyzed. Results: There were 116 patients from eight centers. Seizure onset was in the first year of life in 82%, and mean age at first surgery was 15.8 months (1–35 months). Second surgeries were done in 27 patients, and a third surgery in 6. Etiologies were malformations of cortical development (57), tumor (22), Sturge-Weber syndrome (19), infarct (8), and other (10). Surgeries comprised 40 hemispheric operations, 33 cortical resections, 35 lesionectomies, 7 temporal lobectomies, and one callosotomy. There was one surgical mortality. The most common surgical complications (151 operations in 116 patients) were infection (17) and aseptic meningitis in 13. Of 107 patients with seizure outcome assessed more than one year postoperatively, 72 (67.3%) were seizure free (Engel I), 15(14%) had >90% improvement (Engel II), 12 had >50% improvement (Engel III), and 8 did not benefit from surgery (Engel IV). Development improved in 55.3% after surgery. Conclusion: Epilepsy surgery in children younger than 3 years of age is relatively safe and is effective in controlling seizures. Very young age is not a contraindication to surgery in children with refractory epilepsy, and early surgery may impact development positively.

Neurosurgical management of intracranial epidermoid tumors in children. Clinical article.

Abstract: Object Epidermoid tumors are benign lesions representing 1% of all intracranial tumors. There have been few pediatric series of intracranial epidermoid tumors reported previously. The authors present their experience in the management of these lesions. Methods The neurosurgical database at the Hospital for Sick Children was searched for children with surgically managed intracranial epidermoid tumors. The patients' charts were reviewed for demographic data, details of clinical presentation, surgical therapy, and follow-up. Ethics board approval was obtained for this study. Results Seven children, all girls, were identified who met the inclusion criteria between 1980 and 2007. The average age at surgery was 11.2 years (range 8–15 years), and the mean maximal tumor diameter was 2.1 cm. Headache was the most common presenting symptom, and 1 tumor was found incidentally. Most patients had normal neurological examinations, but meningism was found in 2 cases. There were 3 cerebellopontine angle lesions, 1 pontom...

Presurgical localization of primary motor cortex in pediatric patients with brain lesions by the use of spatially filtered magnetoencephalography.

Abstract: OBJECTIVE: The objective of this study was to confirm the efficacy of spatially filtered magnetoencephalography for the preoperative localization of primary motor cortex in pediatric patients with focal lesions in the region of the sensorimotor cortex. METHODS: We recorded movement-related magnetoencephalographic activity in 10 pediatric patients (age range, 7-18 years; mean age, 12.5 years) undergoing presurgical evaluation for focal brain lesion resection. Participants made transient movements of the right and left index finger in response to a visual cue. The premovement motor field component in the averaged brain response was localized with a newly developed beamformer spatial filter algorithm. Cortical mapping of motor cortex intraoperatively was conducted in 5 of the 10 patients. RESULTS: The motor field time-locked to electromyography onset was successfully localized to cortical areas corresponding to the hand region primary motor cortex in 95% of cases (9 of 10 from nonlesional hemisphere; 10 of 10 from lesional hemisphere). Intraoperative electrocortical stimulation activated the expected muscles at motor field coregistered cortical source locations in all cases tested (n = 5). Using these methods, we also found that displacement of the sensorimotor cortex by space-occupying tumors did not interfere with the localization of motor cortex. CONCLUSION: We conclude that noninvasive localization of the primary motor cortex can be reliably performed by using spatially filtered magnetoencephalography techniques, which provide a robust and accurate measurement of motor cortical function for the purpose of surgical guidance.

The role of the membrane cytoskeleton cross-linker ezrin in medulloblastoma cells

Abstract: Medulloblastoma is a highly malignant brain tumor that occurs predominantly in children. The molecular pathogenesis of medulloblastoma is under investigation. Previously, we used complementary DNA micro-array analysis to compare patterns of gene expression in medulloblastoma samples versus normal cerebellum. The cytoskeletal protein ezrin was found to be overexpressed in medulloblastoma compared with normal cerebellum, an observation that was further validated by immunohistochemistry and real-time PCR analysis. To assess the role of ezrin in medulloblastoma, we studied ezrin’s role in medulloblastoma migration, invasion, and adhesion. Western blotting and immunofluorescence showed high expression of ezrin in four medulloblastoma cell lines, and ezrin was primarily localized to filopodia. Ezrinspecific small interfering RNA suppressed the formation of filopodia and in vitro migration, invasion, and adhesion. We also used a stably transfected medulloblastoma cell line to study the effect of ezrin overexpression. We showed that high expression of ezrin promotes filopodia formation and in vitro invasion. Finally, athymic mice implanted with ezrin-overexpressing DAOY medulloblastoma cell clones in the cerebellum showed shortened survival compared with controls. These findings suggest that, in addition to other cytoskeletal proteins, ezrin plays an important role in medulloblastoma adhesion, migration, and invasion.

Localization of epileptic foci in children with intractable epilepsy secondary to multiple cortical tubers by using synthetic aperture magnetometry kurtosis

Abstract: Object Magnetoencephalography (MEG) has been typically used to localize epileptic activity by modeling interictal activity as equivalent current dipoles (ECDs). Synthetic aperture magnetometry (SAM) is a recently developed adaptive spatial filtering algorithm for MEG that provides some advantages over the ECD approach. The SAM-kurtosis algorithm (also known as SAM[g2]) additionally provides automated temporal detection of spike sources by using excess kurtosis value (steepness of epileptic spike on virtual sensors). To evaluate the efficacy of the SAM(g2) method, the authors applied it to readings obtained in children with intractable epilepsy secondary to tuberous sclerosis complex (TSC), and compared them to localizations obtained with ECDs. Methods The authors studied 13 children with TSC (7 girls) whose ages ranged from 13 months to 16.3 years (mean 7.3 years). Video electroencephalography, MR imaging, and MEG studies were analyzed. A single ECD model was applied to localize ECD clusters. The SAM(g2) ...

Management of pediatric brainstem cavernous malformations: experience over 20 years at the hospital for sick children.

Abstract: Object Because of their location and biological behavior, brainstem cavernous malformations (CMs) pose a formidable clinical challenge to the neurosurgeon. The optimal management of these lesions requires considerable neurosurgical judgment. Accordingly, the authors reviewed their experience with the management of pediatric brainstem CMs at the Hospital for Sick Children. Methods The authors performed a retrospective chart review of pediatric patients who had received diagnoses of a brainstem CM at the Hospital for Sick Children over the past 20 years. Results Twenty patients were diagnosed with brainstem CMs. The mean age at diagnosis was 10.1 ± 5.4 years, and the patients included 13 boys and 7 girls. The mean maximal diameter of the CM was 14.3 ± 11.2 mm. The lesions were evenly distributed on the right and left sides of the brainstem with 4 midbrain, 13 pontine, and 3 medullary lesions. Seven patients underwent surgery for the management of their CMs, with a mean age at presentation of 5.2 years, and ...

Pathology type does not predict language lateralization in children with medically intractable epilepsy.

Abstract: Summary Purpose: We examined potential differences in the effects of pathology type on language lateralization in pediatric epilepsy. Methods: We examined findings from intracarotid sodium amobarbital procedure (IAP/Wada) in a large consecutive sample of children with refractory epilepsy. Subjects were assigned to one of three pathology groups: developmental (n = 28), acquired (n = 26), and tumor (n = 20); groups were compared for language lateralization. Results: Rates of atypical language lateralization did not differ across groups. Greater than half of the subjects with left hemisphere insults and seizure onset before 6 years of age had atypical language lateralization, independent of pathology type. Discussion: Atypical language lateralization may occur in the context of developmental, acquired, and/or tumor pathology.

The role of surgery in the management of intracranial gliomas: current concepts.

Abstract: The role of surgery in the management of human gliomas has been controversial. The results from numerous neurosurgical series are inconsistent. The current adjuvant therapies have facilitated treatment of patients, and have rendered neurosurgical removal without morbidity or mortality more commonplace than ever before. Here, we investigated the role of surgery in the management of adults with low- and high-grade gliomas. Even though there is substantial evidence which claims that surgery per se has a role to play in extending patient survival, there is a paucity of randomized clinical trials on this subject, and little in the way of Class II data to support these claims. However, this should not divert patients away from surgery, because there may be additional benefits from a concerted effort to remove a tumor completely. At the present time, it seems best that clinicians continue to individualize patient treatment based on a myriad of factors that relate to the patient, the patient's tumor, and the known biology of the disease.

Intraoperative arachnoid and cerebrospinal fluid sampling in children with posterior fossa brain tumors.

Abstract: OBJECTIVE: This study was conducted to determine whether arachnoid tissue or cerebrospinal fluid (CSF) sampling is valuable for risk stratification in children with posterior fossa brain tumors. METHODS: Arachnoid tissue and CSF from the cisterna magna (CSF CM ) was sampled at the time of primary tumor resection. Results were compared with conventional staging methods (M stage) and correlated with patient outcome. RESULTS: Eighty-three patients were enrolled in the study. Arachnoid infiltration was identified in 11 of 80 (13.8%) and CSF CM was positive in 20 of 77 (26.0%) specimens. Arachnoid infiltration and CSF cytology were found in 20.0% and 44.8%, respectively, for medulloblastoma/pineoblastoma (primitive neuroectodermal tumor), 6.9% and 3.6% for pilocytic astrocytoma, and 0.0% and 33.3% for ependymoma. The 3-year event-free survival (EFS) was negatively influenced by either arachnoid infiltration (40.9% arachnoid positive versus 65.4% arachnoid negative; P = 0.23) or CSF CM positivity (52.6% CSF CM positive versus 67.1 % CSF CM negative; P = 0.03). The 3-year EFS for patients with primitive neuroectodermal tumor who had positive arachnoid sampling was 33.3%, compared with 67.3% in patients who had no evidence of arachnoid infiltration (P = 0.26). The 3-year EFS for patients with primitive neuroectodermal tumor who had positive CSF CM was 50.0% compared with 67.5% in patients who had negative cytological analysis of CSF CM (P = 0.07). Arachnoid infiltration and CSF sampling were congruous with M stage in 73.3% and 86.2% of patients, respectively. CONCLUSION: Intraoperative evidence of arachnoid infiltration or CSF CM dissemination in patients with posterior fossa brain tumors occurs at a variable frequency that is dependent on tumor type, correlates with conventional M stage, and may be predictive of outcome.

Changing ictal-onset EEG patterns in children with cortical dysplasia.

Abstract: Purpose Cortical dysplasia (CD) is intrinsically epileptogenic. We hypothesize that CDs clinically emerging in the early developing brain tend to extend into multifocal or larger epileptic networks to pronounce intractability in contrast to CDs which clinically emerge at a later age. Methods We evaluated the spatial and temporal profiles of ictal-onset EEG patterns in children with histopathologically confirmed CD. We designated Group A as children with changing ictal-onset EEG patterns over time, and Group B without change. We compared seizure profiles, consecutive scalp video-EEGs (VEEGs), MRI, MEG, and surgical outcomes. Results We found 14 children consisting of 10 Group A patients (7 girls) and 4 Group B patients (all boys). Eight (80%) Group A patients had their seizure onset p Conclusion Ictal-onset EEG patterns change over time in children with early seizure onset and intractable epilepsy caused by CD. Younger epileptic children with CD more frequently have multifocal epileptogenic foci or larger epileptogenic foci. Early resection of CD, guided by MRI, MEG, and intracranial video EEG, resulted in seizure freedom despite changes in ictal-onset EEG patterns.

An intracranial leiomyosarcoma in a child with neurofibromatosis type 1.

Abstract: widespread abnormalities in the nervous system, skin and bones. Neurofibromatosis type 1 (NF1 or von Recklinghausen’s disease) is the most common form, with an incidence of 1/35001. Neurofibromatosis type 1 is a disorder with variable phenotypic expression. Some patients may primarily have cutaneous expression, while others may have life-threatening or severely disfiguring complications. The variability in phenotypic expression of this disease is even demonstrated within families. The NF1 gene is located on chromosome 17 and encodes the tumor suppressor gene neurofibromin, a negative regulator of the Ras signaling pathway2. Inactivation of neurofibromin leads to cell proliferation and tumor development. Neurofibromatosis type 1 is associated with both benign and malignant tumours. The most characteristic tumour in NF1 is the neurofibroma, a nerve sheath tumour composed of schwann cells, fibroblasts, mast cells and vascular components3. The risk of malignancy is three-five times greater in NF1 patients than in the general population4. About 30% of the mortality associated with NF1 is due to a malignancy of some kind. Some of the malignancies associated with NF1 include pheochromocytoma, leukemia, melanoma, carcinoid, Wilm’s tumour and soft tissue sarcomas such as rhabdomyosarcoma and fibrosarcoma. Peripheral nervous system (PNS) tumors such as the plexiform neurofibroma and the malignant peripheral nerve sheath tumour are far more common than central nervous system (CNS) tumours in NF1 patients, but the latter can lead to significant mortality and morbidity. Approximately 90% of the CNS tumours associated with NF1 are pilocytic astrocytomas, the majority of which are optic pathway gliomas1,5,6. In addition to the pilocytic astrocytoma, NF1 patients may develop high grade astrocytic neoplasms. It is estimated that 15% of pediatric NF1 patients will have a brain neoplasm of one kind or another, a statistic that is similar to the adult population7. Since NF1 is a known tumour suppressor gene, its inactivation through a variety of different mechanisms could lead to tumorigenesis including malignant transformation. As such, there are numerous case reports demonstrating the association of NF1 and tumour formation. Some of these tumours, however, may occur with the same frequency in NF patients as they do in the general population. It is therefore important to differentiate these tumours in NF patients from those that are truly associated with NF1 where the incidence in the NF1 population is greater than that of the general population. The present case report depicts a child with NF1 in whom a tumour normally associated with NF1, a leiomyosarcoma, was diagnosed in the intracranial compartment. To our knowledge, this is the first intracranial

The evolution and application of techniques in molecular biology to human brain tumors: a 25 year perspective

Abstract: Since the establishment of the AANS/CNS Section on Tumors in 1984, neurosurgeons have been actively involved in basic science research of human brain tumors that has moved the field forward considerably. Here, we chronicle the major advances that have been made with respect to our understanding of the concepts guiding the biology of human malignant brain tumors. Numerous technical advances in science, such as the development of gene transfer techniques, the polymerase chain reaction, the discovery of oncogenes and tumor suppressor genes, and the refinement of approaches to cancer cytogenetics have enabled researchers to identify many of the non-random genetic alterations associated with brain tumor growth, invasion, immunology, angiogenesis and apoptosis. These data led to some astounding progress, for example with the use of gene therapy, whereby in the 1990s several human clinical trials were conducted for patients with brain tumors. More recently, the human genome project has been completed providing a blueprint for the human species. What has followed are exciting new techniques in molecular biology such as transcriptional profiling, single nucleotide polymorphism (SNP)-arrays, array comparative genomic hybridization (array-CGH), microRNA profiling, and detection of epigenetic silencing of tumor suppressor genes. The cancer genome is now being sequenced at break neck speed using advanced DNA sequencing techniques. We are on the threshold of cataloguing the major genetic alterations observed in all human brain tumors. What will follow is modeling of these genetic alterations in systems that will allow for the development of novel pharmacotherapeutics and translational research therapies.

Dysembryoplastic neuroepithelial tumor

Abstract: The authors1 have succinctly described the course of a 15-year-old boy with dysembryoplastic neuroepithelial tumor (DNET) occupying the corpus callosum and pericallosal region. They have successfully kept the patient symptom free and in remission by surgery alone. Most DNETs are situated in the brain parenchyma, with superficial cortical regions being the most common location. Occasionally, DNETs may be quite aggressive and large, and can extend from the cortex to the white matter, and then to the deep nuclei such as the basal ganglia. Very rarely, malignant features may be seen within these tumors. After 3 years, the patient is doing well without neurological issues. Although the imaging features were suggestive of DNET, the location was not. The authors have done well to draw our attention to atypically presenting DNETs, and to underscore the role of surgery in these cases.

The Guanine Nucleotide Exchange Factor SWAP-70 Modulates the Migration and Invasiveness of Human

Abstract: The malignant glioma is the most common primary human brain tumor. Its tendency to invade away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Accordingly, the molecular pathogenesis of glioma invasion is currently under investigation. Previously, we examined a gene expression array databasecomparinghumangliomastononneoplasticcontrolsandidentifiedseveralRacguaninenucleotideexchange factors with differential expression. Here, we report that the guanine nucleotide exchange factor SWAP-70 has increased expression in malignant gliomas and strongly correlates with lowered patient survival. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac. Using a glioma tissue microarray, we validated that SWAP-70 demonstrates higher expression in malignant gliomas compared with low-grade gliomas or nonneoplastic brain tissue. Through immunofluorescence, SWAP-70 localizes to membrane ruffles in response to the growth factor, epidermal growth factor. To assess the role of SWAP-70 in glioma migration andinvasion,weinhibiteditsexpressionwithsmallinterferingRNAsandobserveddecreasedgliomacellmigrationand invasion. SWAP-70 overexpression led to increased levels of active Rac even in low-serum conditions. In addition, when SWAP-70 was overexpressed in glioma cells, we observed enhanced membrane ruffle formation followed by increased cell migration and invasiveness. Taken together, our findings suggest that the guanine nucleotide exchange factor SWAP-70 plays an important role in the migration and invasion of human gliomas into the surrounding tissue.