Hypertension. Hans-Arne Hansson, Mohamed Al-Ola ma, Eva Jennische, Kliment Gatzinsky, and Stefan Lange .... Sweden; n=90) and female adult albino New Zealand rabbits ..... Hanner P, Rask-Andersen H, Lange S, Jennische E (2010).
The Peptide AF-16 and the AF Protein Counteract lntracranial Hypertension Hans-Arne Hansson, Mohamed Al-Ola ma, Eva Jennische, Kliment Gatzinsky, and Stefan Lange
Abstract Intracranial hypertension develops after, for exarnple, trauma, stroke and brain inflammation, and contributes to increased morbidity, mortality, and persistent neuropsychiatric sequelae. Nonsurgical therapy offers lirnited relief. We investigated whether the peptide AF-16 and the endogenous protein Antisecretory Factor (AF) counteracted abnorma! fluid transfer by cells, and lowered raised intracranial pressure (ICP). Adult rats, infected with an encephalitogenic Herpes simplex virus (HSV-1 ), developed after 5 day s' sickness of increasing severity. AF-16 rescued all rats while vehicle treatment only saved 20%. AF-16 from day 4 reduced the ICP in HSV-1infected rats from 30.7 to 14.6 mmHg and all survived without sequelae. A standardised closed head brain injury in rats raised the ICP. Continuous and intermittent AF-16 kept ICP at an almost normal leve!. A single dose of AF-16 maintained the raised ICP after a TBI lowered during 3-9 h. The AF protein, enriched in egg yolk, sirnilarly lowered the post-traumatically raised ICP in rats. AF-16 also lowered the ICP in rabbits with diffuse brain injury. We conclude that the peptide AF-16 and the AF protein offer new approaches to treat raised ICP with no side effects. Keywords Raised intracranial pressure • Herpes simplex virus encephalitis • Neurotrauma • Sagittal rotational acceleration head and neck irnpulse • Head injury management • Traumatic brain injury • Brain hemiation • Antisecretory factor • S5a
H.-A. Hansson (jgl), E. Jennische, and S. Lange Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, P.O. Box 440, SE 40530 Göteborg, Sweden e-mail: hans-arne.hansson @anatcell.gu.se M. Al-Olama Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, P.O. Box 440, SE 40530 Göteborg, Sweden and Department of Neurosurgery, Sahlgren's University Hospital, SE-41345 Göteborg, Sweden K. Gatzinsky Department of eurosurgery, Sahlgren 's University Hospital, SE-41345 Göteborg, Sweden
lntroduction Raised intracranial pressure (ICP) commonly evolves after a head trauma, stroke, blast, intracranial haemorrhage, brain tumour, inflammation or infection [2 , 5, 16]. Intracranial hypertension disturbs the blood circulation and harnpers the exchange of nutrients, oxygen and metabolites . The tumover of cerebrospinal fluid (CSF) is impaired and the brain parenchyma is concornitantly mechanically distorted and even dislocated by the elevated ICP, especially if pressure gradients develop. The ICP reflects the balance between the close to fixed eraniospinal volume and its ability to accommodate additionally added volume to the incompressible content Swelling of the brain owing to cytotoxic oedema prevails at intracellular fluid accumulation, most evidently in astrocytes [2, ·s, 12, 15, 16] . Leakage from blood vessels, i.e . vasagenie or extracellular oedema, and haemorrhage, increases preferentially the extracellular fluid volume. The volumes of brain parenchyma, blood and CSF may vary dynamically. The cytotoxic and the vasagenie mechanisms are concomitantly operative and thereby their relative contributions to elevated ICP may undergo rapid changes. There is presently no medical treatment that safely and persistently counteracts intracranial hypertension [5, 14, 16]. Treatment with osmotic or hypertonic agents relieves mostly only for a few hours and is often associated with side effects. Surgical measures to improve drainage of the cerebrospinal fluid (CSF) or craniotomy may become necessary as does intensive care [5 , 16, 17] . Improved non-surgical management is thus demanded to keep the ICP sustained at a sufficiently low leve! for extended time periods at no or minimal side effects , beneficial for a large number of victims. The endogenous protein Antisecretory Factor (AF) counteracts and normalises disturbed transfer of fluid through cell membranes and cells [8 , 9, 11, 18] . AF is in addition anti-inflammatory [3, 11 , 18]. A striking observation is that the protein AF and the peptide AF-16 do not affect normal cells and tissues , not even at a high dose [4, 6 , 11 , 18] . The biomedical effects mentioned are exerted by
M.U. Schuhmann and M. Czosnyka (eds.), lntracranial Pressure and Brain M anitaring XIV, . . Acta eurochirurgica Supplementum, Vol. 114, DO!: !0.1007/978-3-7091-0956-4_73, © Spnnger- Verlag/W1en 2012
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an eight amino acid sequence at the amino terminal end of the AF protein [9]. A synthetic peptide, narned AF-16, with the amino acid sequence VCHSKTRSNPENNVGL, which includes the active antisecretory motive of the AF protein, has been demonstrated to be stable and suitable for experimental work. The aim of the present study was to evaluate effects of AF-16 on raised ICP evolving after brain inflammation and an experimentally induced traumatic brain injury (TBI). Time windows for the ICP lowering effects by AF-16 and AF will be discussed. Further, differences in effects of AF and AF-16 on injured brains will be elucidated.
Materials and Methods Animals Mal e Sprague-Dawley rats, 190 ± 20 g, (B & W AB , Stockholm, Sweden; n=90) and female adult albino New Zealand rabbits (2.3-3 kg b. w.; n= 15; local breeder) were purchased. Care was taken to keep the number of animals as low as possible and to minimise their suffering. Permission was granted by the Regional Animal Experiments Ethical Committee and the experiments performed in accordance with local and national guidelines (EU 86/609/EEC).
Chemicals The peptide AF-16 (VCHSKTRSNPENNVGL) [9] was synthesised and characterised by Ross Pedersen A/S, Copenhagen, Denmark. The AF protein was supplied as freeze dried egg yolk (B221 ®; Lantmännen AS-Faktor AB , Stockho1m, Sweden) and manufactured from eggs from hens that had received special feed [ 11]. Isoflurane was purchased from Baxter, and all other chemicals from Sigma-Aldrich.
Treatment with AF-16, AF Protein and SPC Osmotic minipumps (Alzet® 2001 or 2002), containing AF-16 in phosphate buffered saline (PBS) or just the vehicle, PBS, and prestarted, were implanted subcutaneously in the back of rats for continuous delivery for several day s. Further, AF-16 in PBS was either instilled intranasally (i.n.) twice daily or infused intravenously at doses and times indicated. The AF protein, supplied as B221 ®, was dissolved in the drinking fluid and supplied to rats ad libitum.
Herpes Simplex Virus Type l Encephalitis Anaesthetised rats were infected by nasal instillation of 25 J..LL of a diluted solution of an encephalitogenic Herpes simplex virus type I (HSV-1) strain, # 2762, originally isolated from a human fatal case of herpes simplex encephalitis (HSE). Matehed normal rats received an equal volume of the vehicle, PBS . The animals were sacrificed when distinct symptoms of neurological dysfunction, i.e. HSE, appeared. From either day l or day 4 AF-16 in 25 J..LL PBS was instilled twice daily in the right nostril under deep isoflurane anaesthesia, while additional rats had the vehicle, 25 J..LL PBS. The ICP was measured with aminiature light optical system (Samba 3200; Samba Sensors AB, V. Frölunda, Sweden) in anaesthetised rats for at !east l h from day 3-14 [7, 10]. In most experiments two pressure sensors were implanted in parallel, one in each hemisphere. Brains were eventually fixed in 4% formaldehyde in huffered saline and then processed for immunohistochemical investigations to map prevalence and distribution of HSV-1 antigen or for histopathological analysis of stained thin sections [7]. The distribution of vira! DNA was investigated and real-time PCR analyses performed to reveal whether AF-16 exerted any effects.
FocaiBrainlnjury Rats, anaesthetised by isoflurane inhalation, had the calvarium incised and freed from adherent tissue. A copper rod (144 g) with a tip diameter of 4 mm, immersed in liquid nitrogen, was then applied to the right parietal bone for 30 s, inducing a brain tissue necrosis measuring 3 mm in diameter with a depth of -l mm. Great care was taken to keep the exposure conditions reproducible. All animals survived and none showed any obvious signs of irnpairment. The weight gain was checked and found to be the same as for littermates not subjected to any treatment, except for the day of surgery. The extent of brain damage was visualised with 2,3 ,5-triphenyl tetrazolium chloride (TIC), which stains living tissue red while non-vital areas were pale and unstained. The ICP was measured as described above.
Diffuse Bra in Injury Rabbits were exposed to a sagittal rotational acceleration trauma to the head and neck, which induced a diffuse brain injury [10]. The ICP increased in a day and the response to AF-16 was then determined.
The Peptid e AF-16 and the AF Protein Counteract lntracranial Hypertension
Results Normal/CP The ICP in normal adult rats was 5.3±2.1 rnrnHg (n= 15), and vari ed with the exact position of the head and thorax and with age (Fig. la). No rhythrrUcally appearing ICP peaks, either single or in clusters, were demonstrable. Treatment of normal adult rats with 25 f..lg AF-16 intranasally neither induced any significant alteration of the ICP, nor any side effects.
HSV-1 lnfected and AF-16 Treated Rats The treatment of HSV-1 infected rats with l f..lg AF-16 intranasally (i.n.) twice daily for 2 weeks increased the survival rate from 14% for those only having had the vehicle to 40% (p
