BRIEF COMMUNICATION
Complement C5-deficient mice are protected from seizures in experimental cerebral malaria *Susan C. Buckingham, †Theresa N. Ramos, and †Scott R. Barnum Epilepsia, 55(12):e139–e142, 2014 doi: 10.1111/epi.12858
SUMMARY
Dr. Susan C. Buckingham is a postdoctoral fellow in the Department of Neurobiology at University of Alabama at Birmingham.
Studies have demonstrated that the membrane attack complex (MAC) of complement can evoke seizures when injected directly into rodent brain. In the course of studies that examine the role of complement in the development of experimental cerebral malaria (ECM), we observed fewer seizures in mice deficient in C5, a component required for MAC formation. To determine if the MAC contributed to the tonic–clonic seizures characteristic of ECM, we performed long-term video–electroencephalography (EEG) on C5 / mice with Plasmodium berghei ANKA-induced cerebral malaria and observed significantly reduced spike and seizure frequency compared to wild-type mice. Our data suggest a role for the MAC in malaria-induced seizures and that inhibition of the terminal complement pathway may reduce seizures and seizure-related neurocognitive deficits. KEY WORDS: Plasmodium berghei ANKA, Terminal complement pathway.
Malaria remains one of the most common and deadly parasitic diseases worldwide, despite effective chemotherapeutics and widespread distribution of insecticide-treated bednets. The majority of malaria cases are caused by Plasmodium falciparum or Plasmodium vivax; however, P. falciparum causes cerebral malaria (CM), the most severe and frequently fatal form of malaria. Cerebral malaria is distinguished by the development of unarousable coma and occurs primarily in early childhood in endemic regions, although travelers to endemic regions may also be affected.1 Children who develop CM have seizures of a generalized nature, although seizures with a focal origin are frequently observed. Status epilepticus is common in children with CM Accepted September 30, 2014; Early View publication November 10, 2014. Departments of *Neurobiology and †Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Address correspondence to Scott R. Barnum, Department of Microbiology, University of Alabama at Birmingham, 845 19th St. S., Birmingham, AL 35294, U.S.A. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
with concurrent increase in intracranial pressure. Children who survive CM often develop epilepsy and neurologic sequelae, which include transient or prolonged neurocognitive deficits such as psychosis, ataxia, and language and behavioral problems.2 In the course of studies designed to determine the role of the complement system in a well-established animal model for cerebral malaria (experimental cerebral malaria, ECM), we anecdotally observed that mice deficient in complement component C5 had few seizure-like events compared to wild-type mice. In addition, we found that mice treated with anti-C9 antibodies, which prevents formation of the complement membrane attack complex (MAC), had significantly higher survival rates and lower clinical signs of disease,3,4 including seizures. These results suggested that the MAC (which is composed of C5b, a proteolytic fragment of C5, C6, C7, C8, and multiple C9 molecules) may directly or indirectly contribute to the development of malaria-related seizures. To address this possibility, we performed electroencephalography (EEG) studies in which we compared epileptic events between wild-type and
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e140 S. C. Buckingham et al. C5 / mice, the latter of which lack a central component required for MAC formation. For these experiments, mice (seven wild-type and eight C5 / , 8–12 weeks of age, C57BL/6J appropriately backcrossed from our own colony at University of Alabama at Birmingham) were anesthetized and maintained with 2.5% isoflurane. Six small holes were drilled through the skull, bilaterally, approximately 2–3 mm posterior and lateral to bregma, approximately 4 mm posterior to bregma and 5 mm lateral to midline, and approximately 6 mm posterior to bregma and 3–4 mm lateral to midline, using a dental drill equipped with a 1.0 mm drill bit. Three 1.6 mm stainless steel screws (Small Parts, Inc., Logansport, IN, U.S.A.) were screwed halfway into the two holes closest to bregma and in one hole farthest from bregma. Then, an EEG electrode (Plastics One, Inc., Roanoke, VA, U.S.A.) with two lead wires and a ground wire, cut to a length that would touch but not penetrate the dura (approximately 1.5 mm), were inserted into the remaining three drill holes with the ground wire positioned in one of the holes farthest from bregma. The lead wires were placed bilaterally over the cortical surface of the parietal hemispheres in the region over the underlying hippocampi. This two-electrode system does not allow identification of the anatomic origin of epileptic activity. Once the electrode was positioned, dental acrylic was applied to form a stable cap on the skull that cements A
the electrode in place. When the acrylic had dried, the scalp was closed with skin glue (3M Vetbond, St. Paul, MN, U.S.A.). One week after electrode placement surgery, mice were housed individually in specially constructed EEG monitoring cages. EEG data were acquired using Biopac Systems amplifiers (Biopac EEG100C) and AcqKnowlege 4.2 EEG Acquisition and Reader Software (BIOPAC Systems, Inc., Goleta, CA, U.S.A.). Data were stored and analyzed in digital format. Cages were also equipped with IR Digital Color CCD cameras (Digimerge Technologies, Wilsonville, OR, U.S.A.) to record each animal concurrently with EEG monitoring; recordings were acquired for review using security system hardware and software (L20WD800 Series; Lorex Technology, Inc., Markham, ON, Canada). All collected data were manually analyzed for abnormalities by an experienced observer blinded to genotype. All EEG recordings were scored for the presence of isolated spikes, repetitive spiking, and seizures. Spikes were defined as having a duration of
Complement C5-deficient mice are protected from seizures in experimental cerebral malaria *Susan C. Buckingham, †Theresa N. Ramos, and †Scott R. Barnum Epilepsia, 55(12):e139–e142, 2014 doi: 10.1111/epi.12858
SUMMARY
Dr. Susan C. Buckingham is a postdoctoral fellow in the Department of Neurobiology at University of Alabama at Birmingham.
Studies have demonstrated that the membrane attack complex (MAC) of complement can evoke seizures when injected directly into rodent brain. In the course of studies that examine the role of complement in the development of experimental cerebral malaria (ECM), we observed fewer seizures in mice deficient in C5, a component required for MAC formation. To determine if the MAC contributed to the tonic–clonic seizures characteristic of ECM, we performed long-term video–electroencephalography (EEG) on C5 / mice with Plasmodium berghei ANKA-induced cerebral malaria and observed significantly reduced spike and seizure frequency compared to wild-type mice. Our data suggest a role for the MAC in malaria-induced seizures and that inhibition of the terminal complement pathway may reduce seizures and seizure-related neurocognitive deficits. KEY WORDS: Plasmodium berghei ANKA, Terminal complement pathway.
Malaria remains one of the most common and deadly parasitic diseases worldwide, despite effective chemotherapeutics and widespread distribution of insecticide-treated bednets. The majority of malaria cases are caused by Plasmodium falciparum or Plasmodium vivax; however, P. falciparum causes cerebral malaria (CM), the most severe and frequently fatal form of malaria. Cerebral malaria is distinguished by the development of unarousable coma and occurs primarily in early childhood in endemic regions, although travelers to endemic regions may also be affected.1 Children who develop CM have seizures of a generalized nature, although seizures with a focal origin are frequently observed. Status epilepticus is common in children with CM Accepted September 30, 2014; Early View publication November 10, 2014. Departments of *Neurobiology and †Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Address correspondence to Scott R. Barnum, Department of Microbiology, University of Alabama at Birmingham, 845 19th St. S., Birmingham, AL 35294, U.S.A. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
with concurrent increase in intracranial pressure. Children who survive CM often develop epilepsy and neurologic sequelae, which include transient or prolonged neurocognitive deficits such as psychosis, ataxia, and language and behavioral problems.2 In the course of studies designed to determine the role of the complement system in a well-established animal model for cerebral malaria (experimental cerebral malaria, ECM), we anecdotally observed that mice deficient in complement component C5 had few seizure-like events compared to wild-type mice. In addition, we found that mice treated with anti-C9 antibodies, which prevents formation of the complement membrane attack complex (MAC), had significantly higher survival rates and lower clinical signs of disease,3,4 including seizures. These results suggested that the MAC (which is composed of C5b, a proteolytic fragment of C5, C6, C7, C8, and multiple C9 molecules) may directly or indirectly contribute to the development of malaria-related seizures. To address this possibility, we performed electroencephalography (EEG) studies in which we compared epileptic events between wild-type and
e139
e140 S. C. Buckingham et al. C5 / mice, the latter of which lack a central component required for MAC formation. For these experiments, mice (seven wild-type and eight C5 / , 8–12 weeks of age, C57BL/6J appropriately backcrossed from our own colony at University of Alabama at Birmingham) were anesthetized and maintained with 2.5% isoflurane. Six small holes were drilled through the skull, bilaterally, approximately 2–3 mm posterior and lateral to bregma, approximately 4 mm posterior to bregma and 5 mm lateral to midline, and approximately 6 mm posterior to bregma and 3–4 mm lateral to midline, using a dental drill equipped with a 1.0 mm drill bit. Three 1.6 mm stainless steel screws (Small Parts, Inc., Logansport, IN, U.S.A.) were screwed halfway into the two holes closest to bregma and in one hole farthest from bregma. Then, an EEG electrode (Plastics One, Inc., Roanoke, VA, U.S.A.) with two lead wires and a ground wire, cut to a length that would touch but not penetrate the dura (approximately 1.5 mm), were inserted into the remaining three drill holes with the ground wire positioned in one of the holes farthest from bregma. The lead wires were placed bilaterally over the cortical surface of the parietal hemispheres in the region over the underlying hippocampi. This two-electrode system does not allow identification of the anatomic origin of epileptic activity. Once the electrode was positioned, dental acrylic was applied to form a stable cap on the skull that cements A
the electrode in place. When the acrylic had dried, the scalp was closed with skin glue (3M Vetbond, St. Paul, MN, U.S.A.). One week after electrode placement surgery, mice were housed individually in specially constructed EEG monitoring cages. EEG data were acquired using Biopac Systems amplifiers (Biopac EEG100C) and AcqKnowlege 4.2 EEG Acquisition and Reader Software (BIOPAC Systems, Inc., Goleta, CA, U.S.A.). Data were stored and analyzed in digital format. Cages were also equipped with IR Digital Color CCD cameras (Digimerge Technologies, Wilsonville, OR, U.S.A.) to record each animal concurrently with EEG monitoring; recordings were acquired for review using security system hardware and software (L20WD800 Series; Lorex Technology, Inc., Markham, ON, Canada). All collected data were manually analyzed for abnormalities by an experienced observer blinded to genotype. All EEG recordings were scored for the presence of isolated spikes, repetitive spiking, and seizures. Spikes were defined as having a duration of
