Axonal Injury in Cerebral Malaria

Abstract

Impairment of consciousness and other signs of cerebral dysfunction are common complications of severe Plasmodium falciparum malaria. Although the majority of patients make a complete recovery a significant minority, particularly children, have sequelae. The pathological process by which P. falciparum malaria induces severe but usually reversible neurological complications has not been elucidated. Impairment of transport within nerve fibers could induce neurological dysfunction and may have the potential either to resolve or to progress to irreversible damage. β-amyloid precursor protein (β-APP) immunocytochemistry, quantified using digital image analysis, was used to detect defects in axonal transport in brain sections from 54 Vietnamese cases with P. falciparum malaria. The frequency and extent of β-APP staining were more severe in patients with cerebral malaria than in those with no clinical cerebral involvement. β-APP staining was often associated with hemorrhages and areas of demyelination, suggesting that multiple processes may be involved in neuronal injury. The age of focal axonal damage, as determined by the extent of the associated microglial response, varied considerably within tissue sections from individual patients. These findings suggest that axons are vulnerable to a broad range of cerebral insults that occur during P. falciparum malaria infection. Disruption in axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria. Impairment of consciousness and other signs of cerebral dysfunction are common complications of severe Plasmodium falciparum malaria. Although the majority of patients make a complete recovery a significant minority, particularly children, have sequelae. The pathological process by which P. falciparum malaria induces severe but usually reversible neurological complications has not been elucidated. Impairment of transport within nerve fibers could induce neurological dysfunction and may have the potential either to resolve or to progress to irreversible damage. β-amyloid precursor protein (β-APP) immunocytochemistry, quantified using digital image analysis, was used to detect defects in axonal transport in brain sections from 54 Vietnamese cases with P. falciparum malaria. The frequency and extent of β-APP staining were more severe in patients with cerebral malaria than in those with no clinical cerebral involvement. β-APP staining was often associated with hemorrhages and areas of demyelination, suggesting that multiple processes may be involved in neuronal injury. The age of focal axonal damage, as determined by the extent of the associated microglial response, varied considerably within tissue sections from individual patients. These findings suggest that axons are vulnerable to a broad range of cerebral insults that occur during P. falciparum malaria infection. Disruption in axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria. Cerebral malaria (CM) is a diffuse, potentially reversible, encephalopathy, caused by infection with the protozoan parasite Plasmodium falciparum. CM presents clinically with convulsions and coma, and carries 15 to 20% mortality. The malaria parasite invades and develops within erythrocytes that sequester in the cerebral microvasculature and other vital organs by adhesion to specific endothelial receptors.1Turner GD Morrison H Jones M Davis TM Looareesuwan S Buley ID Gatter KC Newbold CI Pukritayakamee S Nagachinta B White N Berendt A An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration.Am J Pathol. 1994; 145: 1057-1069PubMed Google Scholar, 2Turner GDH Cerebral malaria.Brain Pathol. 1997; 7: 569-582Crossref PubMed Scopus (174) Google Scholar The pathophysiological consequences of severe malaria infection have not been satisfactorily resolved. In particular it is not known how parasitized red blood cells, which remain within the vascular space, influence parenchymal brain function to induce coma and death. Histopathological studies reveal a variety of neurological abnormalities including chromatolysis, neuronophagia, and decreased glial cell numbers.2Turner GDH Cerebral malaria.Brain Pathol. 1997; 7: 569-582Crossref PubMed Scopus (174) Google Scholar, 3Marchiafava E Bignami A On summer-autumnal malaria fevers.in: Malaria and the Parasites of Malaria Fevers. The New Sydenham Society, London1894: 1-234Google Scholar, 4Clark H Tomlinson W The pathological anatomy of malaria.in: Boyd M Malariology. W. B. Saunders, Philadelphia1945: 874-903Google Scholar, 5Spitz S The pathology of acute falciparum malaria.Military Surgeon. 1946; 99: 555-572PubMed Google Scholar These changes may result from several coexisting pathological processes including hypoxia, hypoglycemia, cerebral swelling, hemorrhage, and inflammation. Despite these findings in patients who have died from CM, neurological recovery from CM is usually rapid and the majority of patients who survive make a complete recovery within several days. In common with other neurological conditions such as encephalitis, stroke, or neurodegeneration, the response of the brain to an initial parasite-induced insult may be limited to a small number of common mechanisms that can continue to operate after eradication of parasitized red blood cells and contribute independently. If these can be identified they represent attractive targets for novel neuroprotective therapies in CM that could be used as adjuncts to anti-malarial chemotherapy. Axonal injury (AI) is thought to be a common pathway of cerebral injury that occurs in diseases such as multiple sclerosis6Ferguson B Matyszak MK Esiri MM Perry VH Axonal damage in acute multiple sclerosis lesions.Brain. 1997; 120: 393-399Crossref PubMed Scopus (1342) Google Scholar, 7Trapp BD Peterson J Ransohoff RM Rudick R Mork S Bo L Axonal transection in the lesions of multiple sclerosis.N Engl J Med. 1998; 338: 278-285Crossref PubMed Scopus (3542) Google Scholar and stroke.8Pendlebury ST Lee MA Blamire AM Styles P Matthews PM Correlating magnetic resonance imaging markers of axonal injury and demyelination in motor impairment secondary to stroke and multiple sclerosis.Magn Reson Imaging. 2000; 18: 369-378Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Axonal injury disrupts neural integrity, the distribution of neurosecretory granules, and the transport of enzymes and chemicals involved in the formation of neurotransmitters and substances associated with trophic activity. β-amyloid precursor protein (β-APP) is a protein that is normally transported along the axon, and accumulates at the sites of AI.9Gentleman SM Nash MJ Sweeting CJ Graham DI Roberts GW Beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury.Neurosci Lett. 1993; 160: 139-140Crossref PubMed Scopus (495) Google Scholar Positive β-APP immunostaining of axons may identify axons with reversible structural and biochemical changes.10Umehara F Abe M Koreeda Y Izumo S Osame M Axonal damage revealed by accumulation of β-amyloid precursor protein in HTLV-I-associated myelopathy.J Neurol Sci. 2000; 176: 95-101Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Axonal damage may occur gradually, leaving a window for therapeutic intervention during the early stages.11Povlishock JT Traumatically induced axonal injury: pathogenesis and pathobiological implications.Brain Pathol. 1992; 2: 1-12PubMed Google Scholar, 12Povlishock JT Becker DP Cheng CL Vaughan GW Axonal change in minor head injury.J Neuropathol Exp Neurol. 1983; 42: 225-242Crossref PubMed Scopus (361) Google Scholar In this article we have examined evidence for AI in postmortem brain tissue from adult Vietnamese patients who died from P. falciparum malaria. We established a method to quantify the extent of AI using digital image analysis of APP-staining patterns, and determined whether there was any regional susceptibility to AI and related the extent of AI to clinical disease parameters. To define further the causative mechanisms we have correlated AI with other neuropathological features seen in fatal malaria such as demyelination, hemorrhage, sequestration, and glial responses. Specimens were taken at autopsy from adult Vietnamese patients who had died of severe P. falciparum malaria on the Malaria Research Ward, Center for Tropical Diseases, Ho Chi Minh City, Viet Nam. On admission, the presence of P. falciparum malaria parasites in the peripheral blood was detected by routine parasitological examination in all patients. The patients were part of a large double-blind trial of artemether versus quinine for the treatment of severe malaria in Viet Nam.13Hien TT Day NP Nguyen HP Nguyen TH Tran TH Pham PL Dinh XS Ly VC Ha V Waller D Peto TE White NJ A controlled trial of artemether or quinine in Vietnamese adults with severe falciparum malaria.N Engl J Med. 1996; 335: 76-83Crossref PubMed Scopus (397) Google Scholar Two groups were defined prospectively, CM (n = 28) and non-CM (n = 26). CM was defined according to established World Health Organization guidelines as a Glasgow Coma score of 11 or less during the episode of severe malaria,14orld Health Organization Severe falciparum malaria.Trans R Soc Trop Med Hyg. 2000; 94: S1-S90Abstract Full Text PDF PubMed Google Scholar other causes of unconsciousness having been excluded, (eg, hypoglycemia, meningitis, or other encephalopathy) by clinical, biochemical, and cerebrospinal fluid (CSF) examination. Non-CM patients were those dying from severe malaria without coma, who had a range of clinical features typical of other vital organ system complications. A full autopsy was performed within 24 hours of death with consent from the family. All autopsy and specimen collection protocols were approved by the Ethical and Scientific Committee of the Center for Tropical Diseases. Control cases were from a number of different causes of death collected at postmortem at the John Radcliffe Hospital, Oxford, UK, where specific written consent for retention of brain tissues for research purposes had been given. Sampling protocols and use of control UK tissues was approved by the Central Oxford Research Ethics Committee. The controls were cases in which standard neuropathological examination was normal. These included cases in which no history of neurological illness was recorded (acute and chronic deaths from other causes) and cases in which there was a past history of previous neurological illness that was not the direct cause of death. Ten control cases were age-matched to the malaria cases15Medana IM Mai NTH Day NPJ Hien TT Bethell D Phu NH Farrar J White NJ Turner GDH and injury in the of adult Vietnamese patients with fatal Plasmodium falciparum PubMed Scopus Google Scholar and controls were from patients who died from a varied but were within 24 to hours of death. brain in for a of a brain was performed and taken from areas of the brain including with and These were in and using standard and of areas of the were examined to the pathological features of the and sections for β-APP was used to detect defects in axonal transport in brain in were in and to The sections were in with the to a were with for and in The sections were at in of β-APP in and was by with by and with the system controls sections as from of the Positive controls included brain sections from patients with damage, and coma not of were determined using on control and AI was using a of a method by and SM Graham DI A of and in the of axonal PubMed Scopus Google Scholar tissue sections were using a of focal axonal damage were by in and the areas were and using the image at the of Health and on the at A to cell were from the analysis by the to be detected as The of the tissue was The of axonal damage or was as the of tissue with β-APP staining by the of the in was β-APP was to an of the of AI on the brain as a and to for the different brain analysis was performed using the were using and for multiple were for the purposes of and P was as sections were for other neuropathological features that have been in fatal malaria demyelination, with hemorrhage, with an glial with an protein for and an for sections for and were in and the of the was as of examined of process or of the and parenchymal defined as or of suggesting changes in was of included staining of but to a defined as cells within the cells with a to but within the and or with the In an of microglial to AI was used to the of injury in individual The of axonal injury: for 1997; PubMed Scopus Google Scholar, M axonal injury (AI) associated with an early microglial of a for of and PubMed Scopus Google Scholar were to the clinical of the by and different of β-APP staining were in the of fatal P. falciparum malaria These included staining of axons of parenchymal staining more focal parenchymal and and and staining in neuronal cell of the different could be on the The staining was often seen in axonal that had been In sections this was often seen in axonal or as a along an axonal The of β-APP staining in the axons and the parenchymal staining were not as as that in the focal and often an associated microglial response, suggesting that these were The staining was by the of of the large areas of tissue sections were with of axonal Although the were not the β-APP was to the with was often to AI with other pathological The focal of AI were for The areas of injury were and of to of of axonal that as a of axonal to on axonal and and The number of patients with or focal AI in the brain was in CM patients with non-CM patients of with Axonal in the CM with Non-CM or Control of patients with of patients without P P in a were often associated with of of A and or of secondary demyelination of of and in it was that AI was and the result of In these staining was within the of hemorrhages A and We not an distribution of AI within a there was a AI and vascular = was no significant the presence or of and CM or were not an in of malaria cases parasite infection. The of with individual cells, with large of cells The were but were In no was the presence or of or and CM or and were in and of the malaria was in the age of the AI within individual patients. the AI during the or considerably death could be by the extent of the associated glial response, associated secondary pathological and the extent of AI along the of changes of patients microglial suggesting that the lesions were several included those patients who had of AI lesions with and without associated microglial on the E and was that in patients the AI in different of the in other cases they at different We no focal to AI and and there was no significant and were not in patients with severe of severe malaria patients of in the in the parenchymal and and in the was in patients with severe malaria but was not associated with CM or was a the of and hemorrhages in the = was within and cases in the extent of AI in different brain In it was that AI within brain was The was for brain including and and an for individual was were significant in the regional β-APP staining in the malaria as a with with the of the where no significant was = the of AI in patients dying of CM and those dying of non-CM a of AI in CM in = = and = as as the all brain = there was no significant the groups in the of AI in the = were significant the of AI and in CM patients with controls with the of the = were no significant non-CM and control patients in the of AI for any brain P = P = P = P = These are in of Axonal in CM with Non-CM and Control with with with with The of axonal damage or was as the of tissue with staining by the of the with with with The of axonal damage or was as the of tissue with staining by the of the P with P with P with P with P with The of axonal damage or was as the of tissue with staining by the of the in a To the of these of we performed a the AI and within individual brain and the presence of a number of clinical and biochemical using all malaria patients. These included the to death from and of coma and Glasgow coma of and protein and cell included the number of World Health Organization the of severe malaria in an individual as a of the of and the presence of individual including and acute all these Glasgow coma and to death significant with AI staining or not in all brain examined are in versus on admission, Glasgow coma and death. an individual = of tissue with axons by the of the = of in and of an individual image The with AI was the Glasgow coma score were of coma as by Glasgow coma score and the of the the coma the more in all brain with the of the AI or AI in the and protein were significant AI or AI in the and on and death were of as a of and AI in the and but not in the or AI was as a brain was no of AI with of severe were patients with CM without other organ complications. The AI varied considerably within this is the study of P. falciparum malaria to a the clinical of system infection and the mechanisms of neurological injury during a that could the of neurological dysfunction with the potential to resolve or progress to irreversible damage during severe malaria infection. In to previous of cerebral and injury IM Mai NTH Day NPJ Hien TT Bethell D Phu NH Farrar J White NJ Turner GDH and injury in the of adult Vietnamese patients with fatal Plasmodium falciparum PubMed Scopus Google Scholar the and extent of AI the groups of patients with P. falciparum with and without neurological complications during The other pathological that in and in previous CM from non-CM in this of patients was the of of parasitized erythrocytes within cerebral In this study hemorrhage, glial and the presence of not the not a of these to the of CM, as these are more and to change within of the of AI for several to changes in system with or glial often for from cell of and may on of to and are to or damage in several different vascular In P. falciparum malaria is The that axons may that is of cell R function and in during 1997; PubMed Scopus Google Scholar may we no with specific of neuronal or glial injury in a of these IM Mai NTH Day NPJ Hien TT Bethell D Phu NH Farrar J White NJ Turner GDH and injury in the of adult Vietnamese patients with fatal Plasmodium falciparum PubMed Scopus Google Scholar to the of of AI in CM Although AI was a significant in the CM the age and distribution of the as as the of AI with other pathological were features individual patients with that the cerebral insults to which axons are during fatal P. falciparum malaria Disruption in axonal transport may represent a common pathway leading to potentially reversible neurological is that AI occurs in patients who survive and from These be in patients using magnetic resonance imaging for an of axonal and from these patients for markers of magnetic resonance imaging are not in AI a potential for neuroprotective in of axonal by β-APP we have detected more changes to neuronal function a of the Although β-APP is a of axons these are not detected by standard immunohistochemical β-APP accumulates at sites of axons within hours after and in axons and for more than SM Nash MJ Sweeting CJ Graham DI Roberts GW Beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury.Neurosci Lett. 1993; 160: 139-140Crossref PubMed Scopus (495) Google Scholar, The of axonal injury: for 1997; PubMed Scopus Google Scholar, SM Graham DI a marker of axonal damage in head 1996; PubMed Scopus Google Scholar In this all fatal malaria cases died within of to all axons remain using this β-APP staining has been to occur in J GW of the precursor protein in PubMed Scopus Google Scholar but we different staining with and β-APP E and To define further the mechanisms of AI we have multiple of the sections for other pathological and correlated AI with other clinical and biochemical parameters. Two of AI from this The a of all brain The a of AI in brain the or may different The potentially an insult multiple brain such as that which could be after a organ or vascular In the brain may a In a previous study it was that cases of CM from Vietnamese of within different areas of the Y M of parasitized erythrocytes in the and in cerebral J Trop Med Hyg. 1993; Google Scholar In to the AI the cerebral and were to or the that AI is not related to the extent of of β-APP staining is but by no specific for a particular of injury. of disease and F Abe M Koreeda Y Izumo S Osame M Axonal damage revealed by accumulation of β-amyloid precursor protein in HTLV-I-associated myelopathy.J Neurol Sci. 2000; 176: 95-101Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, F Esiri MM Cerebral damage in infection using precursor protein 1997; PubMed Scopus Google Scholar, F L F F L F R brain with and axonal damage in the 1998; PubMed Scopus Google Scholar the distribution of AI was not to a distribution within the of not the of that are not in the in conditions is and correlated with acute neuronal Perry VH on cerebral the of a or during acute neuronal PubMed Scopus Google Scholar there was no significant the presence or of and CM or AI was of hemorrhage, and glial responses. the was not of microglial were they an of the of the axonal In lesions to axonal within the brain in other patients multiple lesions at different within the brain as by the different microglial responses. lesions within the in sites of the brain in other cases brain to have been considerably These findings further the and that has been in other studies of fatal GDH Cerebral malaria.Brain Pathol. 1997; 7: 569-582Crossref PubMed Scopus (174) Google Scholar, 3Marchiafava E Bignami A On summer-autumnal malaria fevers.in: Malaria and the Parasites of Malaria Fevers. The New Sydenham Society, London1894: 1-234Google Scholar, 4Clark H Tomlinson W The pathological anatomy of malaria.in: Boyd M Malariology. W. B. Saunders, Philadelphia1945: 874-903Google Scholar, 5Spitz S The pathology of acute falciparum malaria.Military Surgeon. 1946; 99: 555-572PubMed Google Scholar has the that B The role of in the formation of axonal Pathol. PubMed Scopus Google Scholar B The role of in the formation of axonal Pathol. PubMed Scopus Google Scholar, Graham DI B The of the after an acute brain 2000; PubMed Scopus Google Scholar, DI D damage in head injury secondary to PubMed Scopus Google Scholar, D Graham DI is an cause of axonal 2000; PubMed Scopus Google Scholar and D Graham DI is a cause of axonal 2000; PubMed Scopus Google Scholar can cause We no AI and or the and is not defined in there was no with impairment of vital organ function such as or was no of AI with of of which may that these patients are and more to the extent of AI within the system is determined or early in the there were with and Glasgow coma is with an of axonal damage in who had reversible as as in those with more severe associated with head lesions in the brain head injury.J Neurol PubMed Scopus Google Scholar that axonal damage may be associated with clinically reversible coma in and malaria. AI was in the of patients who died within of to the patients with CM without other organ the AI was the that AI can occur without other of severe but that not with clinical in in part of the of ST Lee MA Blamire AM Styles P Matthews PM Correlating magnetic resonance imaging markers of axonal injury and demyelination in motor impairment secondary to stroke and multiple sclerosis.Magn Reson Imaging. 2000; 18: 369-378Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar In we have a marker of potentially reversible axonal damage that is associated with the of CM in adults and that CM from non-CM patients. marker the and as areas of in other pathological such as neuronal AI not to the of severe malaria to the specific neurological of the mechanisms of axonal is an in the specific neurological complications associated with We the Central Oxford Research Ethics Committee and the of patients included for to these the of the Malaria Research Ward, Center for Tropical Diseases, Ho Chi Minh City, for to this the and of the Center for Tropical for and for this and the of the The John Radcliffe Hospital, Oxford, for with of