S267

Oral Presentations

Friday, 11 July 1997

0-30 Awardees' Presentations

09:00-14:00

10-30-4391 GrC?~h inhi~itory and differentiation inducingactiVity of dlmethylformamide In cultured humanmalignant glioma cells

X.-N. U. SuzhouMedicalCollege, Suznou, PR China

No abstractreceived

I0-30-4381 The role of angiogenesis in the pathogenesis ofdural arteriovenous malformations redefined

M.T. Lawton, R. Jacobowitz, R.F. Spetzler. BarrowNeurological Institute,Phoenix, Arizona, USA

Introduction: Dural arteriovenous malformations (DAVMs) appear to be ac­quired rather than congenital lesions, but their pathogenesis remains unclear.According to the prevailing hypothesis, DAVMsarise from intrinsicarteriove­nouscommunications in the dura materthat opento formfistulae. Alternatively,dysautoregulatory vasodilation in response to venous sinuses into DAVMs.These hypotheses share the notionthat DAVMsarisefrom pre-existing vascu­lar channels in the dura. However, DAVMs could also arise from newly formedbloodvessels (angiogenesis) as part of the inflammatory processthat recanal­izes thrombosed sinuses. These hypotheses are supported by few laboratorydata. Rat modelshave confirmed thaI venous hypertension and sagittal sinusthrombosis elicit Ihe formation of DAVMs. This study providesthe first experi­mentalevidencesupporting angiogenesis as a mechanism of DAVMformationand implicatesa crucial role for cerebral ischemia, rather than inflammation, inthe process.

Methods: Forty rats underwent common carotid artery-te-extemal jugu­lar vein (CCA-EJV) anastomosis, bipolar coaquanon of the vein drainingthetransverse sinus,and sagittalsinusthrombosis to inducevenoushypertension.Fifteen rats underwent a similar surgical procedure, but venoushypertensionwas not induced. The 55 rats were divided into seven groups. Four groupseachcontained 10 rats withvenoushypertension. The otherthreegroupseachcontained five rats without venous hypertension. After t, 2, or 3 weeks, thedura from one group each of the hypertensive and nonhypertensive rats wasassayedfor angiogenic actiVity (rabbit comea bioassay). The remaining groupof 10hypertensive ratswasnotassayedto determine if sampling affected DAVMformation. All ratssurvivedfor 3 months, after whichcerebral angiography wasperformed to assessDAVM formation.

Results: Unlike rats without CCA-EJV anastomosis, rats with CCA·EJVanastomosis had significantly increased postoperative sagittalsinuspressures(p < 0.0001). Mean angiogenesis indices were significantly greater in ratswithvenoushypertension than in ratswithoutvenoushypertension (p = 0.004).Angiogenic activitywas greatest at 1 week and decreased over time. DAVMSformed in 42% of the 55 rats; none of the rats without venous hypertension(Spearman rank correlation coefficient =0.74). Development of DAVMs cor­related positivelywith both venous hypertension (p =0.0009) and angiogenicactivity (p =0.04).

Conclusion: Significant relationships between venous hypertension andangiogenic activity and between angiogenic actiVityand DAVM formation havebeen demonstrated. These results suggest a new angiogenic hypothesis ofDAVMformation. Briefly, venoushypertension inducedby an obstruction to ve­nousoutflowsuch as sinus thrombus initiatesthe pathogenesis of DAVMs. Ve­noushypertension reducescerebral perfusion and produces cerebral ischemia.Tissue hypoxia normally stimulates angiogenesis in an attempt to reverse is­chemia, and the ischemia induced by venous hypertension would also elicitangiongenesis. Humoral factors released by ischemic brain adjacent to thedural sinuses might affect meningeal capillaries, which might connect to duralsinusesto initiate arteriovenous shunting. Therefore, aberrant angiogenic ac­tivityby duralblood vesselswould lead to arteriovenous shunting into the duralsinusesand DAVM formation. Arterialization of the dural sinusesexacerbatesthe undertying venous hypertension; turbulentblood flow exacerbates the un­derlyingvenousoutflowocclusionby injuringintima,thickening sinuswalls,andpromoting lumenal thrombosis.A vicious cycle would thereby be created thatwouldamplifythe hemodynamics initiating DAVMformation, promote continuedangiogenic activity, enlarge the DAVM, and lead to a more dangerous clinicalcourse.

I0-30-440 IThe effects of chronic cerebral hypoperfusionassociated with arteriovenous malformations

L.H.S. Sekhon. TheUniversity of Sydney, Sydney, Australia

Introduction: The adverseeffectsof cerebral ischemiawere recognised sincethe time of Hippocrates and although over two-thousand years have passed,~~Iy recently havewebegunto understand the mechanisms leadingto neuronalInjuryand cell death. Our knowledge of the effects and mechanisms of acuteischemia is improving, though little is known about the effects of reductionsin cerebral blood flow (CBF) in the chronic hypoperfusion. Acute reductions inCBF of less than 50% do not affect neuronal structureor function. The effectsof chronicreductions in CBFof up to 50% suchas thoseoccurringsecondary toartenovenous steal have not been previously explored. This aim of this seriesof experiments was to evaluate sequentially in vitro structureand functionandthen in vivo animalbehaviorin order 10 ascertain what were the effects, if any,occurred witha chronicreduction ofCBFbetween25 and50% maintained for26weeks. Thus it wouldbe hopedthat in conclusion that a greaterunderstandingcould be attainedof the centralnervoussystemchangesthat occur in chroniccerebral hypoperfusion in general, and morespecificallyin the association withcerebral arteriovenous malformations (AVMs).

Materials and methods: Male Sprague-Dawley rats (250-300 g initialweight) underwent general anaesthesia (1.6% halothane) and had an arteri­oven?u~ fistula created surgically in the neck. Under direct magnification acarotid-Jugular fistula was created by caUdally ligating the right internal carotidartery and extemal jugUlar vein and forming an end-to-end anastomosis be­tweenthe common carotidarteryand the externaljugularvein with interrupted10-0 nylon [1--4). This formed a functional arteriovenous fistula between theanterior intracranial circulation and extracranial venous circulation and thuscreatedcerebralhypoperfusion withoutan acute ischemicinsult.CBF(as mea­suredvia 14Cau,t0radiography) was found to be reduced by 25-50%, creatinga modelof chroniccerebral hypoperfusion in the absenceof an acute ischemicinsult.Age-matched animalswereusedas controls.

Six monthsafter fistula formation, the animalswere examined in one of 3experimental regimes:(1) In vitro structuralassessment Histopathological assessmen1 of the hip­

pocampus usmqlight (LM)and electron microscopy (EM)(2) In vitrofunctional assessment: Electrophysiology in the hippocampus utilis­

ing invitrohippocampal fieldpotentialrecording to examinevariousfacetsoflongterm potentiation (LTP) in the hippocampus between controland fistulaanimals

(3) In vivofunctional assessment: Behavioral testingofanimalsusingopen-fieldstudies, T-maze analysis andmotorexaminationsResults: The LM and EM studies demonstrated an absence of cerebral

infarction on surveyof all brain regions, but there was markeddisorganizationof cell regions in the hippocampus in theanimalswho possessed arteriovenousfistulae, most pronounced in the CA1 region [6]. There was also greatly in­creased tissuevascularity with increased numbersof capillaries, someof whichdid nothaveperiluminal GFAPstaining suggesting an absenceofastrocyticfootprocesses 17].

Electrophysiological recording showedthatLTPwas impaired after26 weeksofchroniccerebral hypoperfusion,5 butwas intactafteronly10weeksofasimilardegreeof ischemia [8].Theinducedchangestook timeto develop. LTPinitiatedthrough o~er ",leans(tr~nsienl hypercalcemia asopposed to tetanicstimulation)was also Impaired. An Increase 10 background inhibition in the hippocampuswas not thoughtto playa role(10).

Behavioral studiessuggested thatthe fistulaanimalshadgreaterexploratorybehavior with a lowered "emotionality", and Impaired reference memory, inparticular its consolidation [6]. No differences in motor functionwere found.

Conclusions: The results presented here show changes quite distinctfrom those seen during either acute cerebral ischemiaor during the recoveryphaseafter the ischemic insult.Chronichypoperfusion with reductions of CBFof 25-50% as may occur in the cerebrovascular steal associated with AVMscan lead to structural and functional neuronal changes and may also causechanges inanimalbehavior. Themechanisms forthesechangesandreversibility

S268 Awardees' Presentations Friday, 11 July 1997

are unknown and the significance of these findings in clinical terms is as yetunclear. The capillary abnormalities may explain the predisposition to normalperfusion pressure breakthrough after AVM resection. Previously it was thoughtthat CBF had to be reduced by more than 50% in order to have deleteriouseffects on neurons. The effects of chronic reductions in CBF had not beenassessed. The pathophysiological processes occurring in patients with largelong-standing AVMs is likely linked to chronic non-infarctional hypoperfusionand in contradistinction to acute ischemia, behavioral and pathological changesmay occur in the absence of infarction. Two major implications, not previouslydescribed, emerge from these experiments with respect to chronic cerebralischemia. Firstly, the thresholds of acute cerebral ischemia deviate from normalin the presence of a chronically maintained ischemic insult. Secondly, a newpathological subtype of chronic cerebral ischemia is defined between the twopolarities of either neuronal normality or neuronal infarction, whereby in vitroneuronal structure and function and in vivo whole animal behavior can becompromised in the absence of cerebral infarction following a chronic reductionin CBF of 25-50%. These experiments thus provide the initial foray into theexploration of a new SUbtype of cerebral ischemia as well as further quantifyingthe pathophysiology associated with cerebral AVMs.

[1] Morgan M.K., Anderson, R.E., Sundt, T.M., Jr. A model of the pathophysi­ology of cerebral arteriovenous malformations by a carotid-jugular fistula inthe rat. Brain Res. 496: 241-250, 1989.

[2] Morgan, MK, Anderson, R.E., Sundt, T.M., Jr. The effects of hyperventilationon cerebral blood flow in the rat with an open and closed carotid-jugularfistula. Neurosurgery 25: 606--612, 1989.

[3] Morgan, M.K., Johnston, I., Besser, M. et al.: Cerebral arteriovenous mal­formations, steal, and the hypertensive breakthrough threshold. An experi­mental study in rats. J. Neurosurg. 66: 563-567, 1987.

[4] Morgan, M.K., Sundt, T.M., Jr., Anderson, R.E.: The haemodynamic conse­quences of a carotid-jugular fislula in the rat during hypocapnoea. J. Clin.Neuroscience 1: 193-196, 1994.

[5] Sekhon, L.H.S., Morgan, M.K., Spence, I. et al.: Chronic cerebral hy­poperfusion: pathological and behavioral consequences. Neurosurgery 40:548--556, 1997.

[6] Sekhon, L.H.S., Spence, I., Morgan M.K.: Normal perfusion pressure break­through: the role of capillaries. J. Neurosurg. 86: 519-524, 1997.

[7] Sekhon, L.H.S., Spence, I., Morgan, M.K. et al.: Chronic celebral hypoper­fusion in the rat: temporal delineation of effects and the in vitro ischemicthreshold, Brain Res. 704-11t, 1995.

[8] Sekhon, L.H.S., Spence, I., Morgan, M.K. et al.: Chronic cerebral hypoper­fusion inhibits calcium-induced longterm potentiation in rats. Stroke 1997 (InPress).

[9] Sekhon, L.H.S., Spence I., Morgan, M.K. et al.: The role of inhibition inchronic cerebral hypoperfusion. J. Clin. Neurosci. 1997 (In Press).

I0-30-441 I Regeneration of the injured acoustic nervepromoted by intrathecal treatment withtheantibody IN-1 against neurite growth inhibitors

M. Tatagiba 1.2, S. Rosahl 2 , C. Brosarnie 1, A. Brandis 3, A. Skerra 4,

M. Samii 2, M.E. Schwab 1. 1Brain Research Institute, Universityof Zurich,Zurich,Switzerland, 2 Departmentof Neurosurgery, NordstadtHospital,Hannover, 3 Institute of Neuropathology, HannoverMedicalSchool, Hannover,4 Departmentof Protein Chemistry, Technische Hochschule, Darmstadt,Germany

Injured axons of peripheral nervous system (PNS) have a high capacity forregeneration, whereas injury to axons in lhe adult central nervous system (SCN)of higher species does not generally result in regeneration. One major reasonfor this difference is the presence in CNS myelin of proteins that inhibit neuritegrowth. These inhibitory proteins (called NI-35 and NI-250) wer shown to beundetectable in the PNS myelin. Neutralization of the inhibitory proteins by amonoclonal antibody (IN-I) directed against the inhibitors led to marked axonalregeneration followed by functional recovery in adult spinal cord lesioned rats.Besides, several neurotrophic factors that have been identified in recent yearswere shown to promote survival ofaxotomized neurons and enhance axonalsprouting in CNS.

Microsurgical crush or cut lesion of the cochlear nerve was performed inadult Lewis rats, which completely internupted the cochlear nerve axons at thelesion site producinq ipsilateral deafness in all animals. The rats were intrathe­cally infused with a recombinant Fab fragment to the antibody IN-l againstmyelin-associated neurite growth inhibitory proteins for one week. An aged­matched control group of rats was treated with unspecific mouse IgG antibody.Because the nerve lesions resulted in significant neuronal apoptosis of primaryauditory neurons of the spiral ganglion, neurotrophin-3 (NT-3) was applied tothe lesion site immediately post-injury in some rats. Electrophysiological studieswere carried out in a group of rats by recording the brainstem auditory evokedpotentials (BAEP) in regular intervals up to 2 months after the injury. Morpho­logical studies of the cochlear nerve axons were done with anterograde tracers

by injecting horseradish peroxidase (HRP) or biotinylated dextran amine (BOA)into the spiral ganglion.

The results obtained in this study were consistent with following conclusions:1) crush injury to the cochlear nerve in the adult rat CPA results in functionaldeafness, disappearance of BAEP, apoptosis of parent axotomized neuronsof the spiral ganglion, and internuption of labeled axons close to the lesionsite; 2) NT-3 is able to partially rescue back-dying axotomized neurons andto increase the number of available fibres; 3) lesioned cochlear nerve fibresshow a limited spontaneous sprouting and regrowth response which do notlead to BAEP recovery; 4) the presence of the antibody IN-t directed againstthe myelin-associated neurite growth inhibitory proteins promotes significantelongation of the injured fibres; and 5) the regenerating fibres navigate to correcttargets. and are capable of establishing synaptic connections for functionalrecovery as depict by BAEP examinations.

I0-30-4421 Overexpression of the growth hormone-releasinghormone genein acromegaly-associated pitUitarytumors: Aneventassociated with neoplasticprogression andaggressive behavior

K. Thapar 1, K.Kovacs 2, L. Stefaneanu 2, B. Scheithauer 3, O. Killinger 4,R.V. Loyd 3, H. Smyth 1, M.O. Thorner 5, B. Gaylinn 5, E.R. Laws, Jr. 6.1 Departments of Neurosurgery and 2 Pathology, Universityof Toronto,Toronto, Ontario, Canada, 3 Departmentof LaboratoryMedicine,MayoClinic,Rochester, MN, USA,4 Departmentof Medicine. Universityof Western Ontario,London, Ontario, Canada, 5 Departments of MedicineandeNeurologicalSurgery(ERL), Universityof Virginia, Charlottesville, VA, USA

The pituitary tumors underlying acromegaly, although unified by their pathologichypersecretion of growth hormone (GH), are an otherwise heterogeneous groupof lesions. From biological, clinical, and prognostic standpoints, the behavior ofthese tumors is highly variable and generally defies reliable prediction. Whereassome somatotroph adenomas are amenable to curative resection, others willprogress relentlessly, often in spite of maximal surgical, pharmacologic, and ra­diotherapeutic intervention. Such variability in tumor behavior, although neitherreflected in nor predicted by the tumor's histologic or ultrastnuctural morphology,is presumably the result of specific subcellular events that promote neoplasticprogression among aggressive variants. To date, however, prognostically infor­mative determinants of neoplastic progression remain poorly characterized inthis tumor system.

A hypothalamic peptide and potent mitogen, growth hormone-releasing hor­mone (GHRH) is the principal physiologic trophic regulator for GH-producingcells (somatotrophs) of the pituitary. Following its release from hypothalamicnuclei and subsequent descent via the portal circulation, GHRH binds to itsreceptor (GHRH-R) on the somatotroph cell surface, stimulating both the pro­liferation of these cells and their secretion of GH. In logical extension of suchtrophic physiologic activity has been the implication that excessive GHRH stim­ulation may have some role in the development and progression of somatotrophtumors. That mice bearing the human GHRH transgene develop GH producingpituitary tumorsprovides compelling evidence in support of such a role, as doesthe occurrence of clinical acromegaly, sornatotroph hyperplasia and/or soma­totroph adenomas in patients exposed to chronic GHRH excess in the setting ofrare ectopic GHRH producing tumors. More recently, there have been accountsof GHRH mRNA transcripts and immunoreactive GHRH being present withintumors cells of human GH-producing pituitary adenomas. Collectively, thesedata raise the intriguing possibility that these tumors may be engaged in lo­cal production of GHRH, generating self-sustaining GHRH mediated autocrineand/or paracrine stimulatory loops, ones capable of driving the progression ofestablished tumors.

In this report, we have demonstrated that tumoral overexpression of thegrowth hormone-releasing hormone (GHRH) gene is one prognostically infor­mative event associated with the clinical aggressiveness of somatotroph pitu­itary tumors. Accumulation of GHRH mRNA transcripts was demonstrated in91 of a consecutive series of 100 somatotroph tumors by in situ hybridization;these findings were corroborated by northern analysis, reverse transcriptasepolymerase chain reaction, and protein translation was confirmed by Westernblotting. By comparison, transcript accumulation was absent or negligibly low in30 normal pituitary glands. Accumulation of GHRH mRNA transcripts was nota random phenomenon; instead transcripts were found to preferentially accu­mulate among clinically aggressive tumors. Specifically, GHRH mRNA signalintensity was: (i) linearly correlated with Ki-67 tumor growth fractions (r = 0.71,P < 0.001); (ii) linearly correlated with preoperative serum GH levels (r = 0.56,P = 0.01); (iii) higher among invasive tumors (p < 0.001); and (iv) highest inthose tumors in which postoperative remission was not achieved (p < 0.001).That the relationship between GHRH overexpression and aggressive clinicalbehavior may have an autocrine and/or paracrine basis was supported by theadditional demonstration on Northern analysis that GHRH and GHRH-receptortranscripts are co-expressed by somatotroph adenomas.

To delineate more precisely the relationship between GHRH mRNA tran­script accumulation and surgical outcome, particularly as compared to other