High molecular weight dextran as a new, safe and reliable additive in cerebral microdialysisÌý
Francesco Fiorini
Supervisor: Dr. DavidÌýJuncker
Trauma can strike everyone, whether performing specifically risky activities or simply conducting our everyday lives. Head trauma in particular, also referred to as traumatic brain injury or TBI, can have extremely catastrophic consequences as the very core of our being is taking the hit: the brain. The intricacy of this fascinating structure is in fact but the sharpest of double- edged swords, earning it the pride of being Nature’s finest creation but at the same time limiting incredibly its healing capacity. Very little can indeed be done to predict the brain’s response to trauma and promote its feeble repairing process, with TBI still representing the single greatest cause of death and disability in young people. But things might be changing, and my chance to contribute to this field presented under a neuroscience research exchange between my medical school at Imperial College London and Â鶹AV, where I joined Prof Juncker’s Micro and NanoBioengineering lab.Ìý
The damage to the brain associated with TBI is of a very peculiar type, in that it comes in three subsequent bouts. Firstly, the traumatic event itself strikes the brain, which will react very specifically according to the bio- psychosocial status of each individual. While measures can be taken to prevent trauma in the first place, primary damage is virtually impossible to revert once occurred. This can then trigger a cascade of molecular processes that contribute to the ignition of progressive damage, a second stage which might affect a much wider area of the brain. Eventually, this will lead to irreversible secondary injury. Progressive damage is thought to evolve over approximately 6 hours from the initial trauma and represents the main and possibly sole window for significant clinical intervention. A number of mechanisms have been found to be involved, deriving predominantly from the lack of oxygen in the affected brain tissue. Each of them will manifest as very specific variations in the concentration of key molecular agents, including for instance raised levels of neurotransmitter glutamate, shifts in metabolic products, and production of inflammatory cytokines. While a number of well-established instruments enable monitoring of crucial parameters following TBI – chiefly intra-cranial pressure, cerebral perfusion pressure and brain oxygenation levels – the detection of specific molecular fluctuations has been made possible only recently with the advent of cerebralÌýmicrodialysis, a unique sampling technique to systematically measure the concentration of target molecules in the interstitial space in vivo (Figure 1).Ìý
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Figure 1 Ìý ÌýÌýSchematic representation of the cerebral microdialysis technique.Ìý
Microdialysis relies on the constant pumping of perfusion fluid into a catheter, reaching a diffusion membrane positioned intracerebrally. Here, small quantities of molecules mirroring the brain’s chemistry diffuse into the perfusate, now termed dyalisate, which keeps flowing, enters the distal part of the catheter and is collected into end micro-vials, ready for analysis. However, the recent introduction of catheters with higher molecular weight cut-off diffusion membranes (100 kDa), i.e. able to collect larger molecules from the interstitial space such as proteins, highlighted a problem in this system. The standard artificial cerebro-spinal fluid (aCSF) perfusate typically used, a saline solution, undergoes a phenomenon known as ultrafiltration due its low tonicity and is seen to effectively leak through the membrane andÌýpotential leakage of these additives through the diffusion membrane, being both smaller than the 100-kDa cut-off membrane pores.
Our research focused on optimising this system using an in vitro model, comparing the known aCSF, serum albumin, and LMW dextran perfusion fluids to a new candidate: 3% high molecular weight (HMW) dextran (200-300 KDa). We showed that there is no significant difference between these fluids in their ability to recover four small key molecules (glucose, pyruvate, lactate, and glutamate), with HMW dextran displaying equal performance as currently used perfusates. Moreover, we found our new candidate to be overall a safer choice, minimising fluid exchange between the catheter and the sampled region and leakingÌýless through the diffusion membrane. In addition to this, dextran is known to be a biologically inert compound with extremely low immunogenicity, while the same cannot be stated for albumin, a protein largely found in the human body and covering a plethora of functions. Furthermore, we strongly advise toward discarding the microdialysate sample retrieved in the first 20 minutes of operation, as the flow instability observed during this initial phase is likely to produce uncontrollable variations on measurements, thus invalidating the collected sample.
Future work will focus on elaborating our data on the recovery of larger proteins, as well as studying the more widespread effect thatÌýthese colloids might have on cultures of neural cells and assessing more systematically their leakage through the diffusion membrane, using radioactively labelled versions of these additives. This will be possible thanks to an additional summer scholarship awarded by the Natural Science and Engineering Research Council of Canada.
Altogether, our results lead us to recommend using 3% HMW dextran as a novel, reliable and safer perfusion fluid in cerebral microdialysis. We believe that the optimisation of this monitoring platform will soon make it a fundamental neuro- physiological instrument in critical care.Ìý
F.Fiorini 1,2,4,5, V. Laforte 2,4,5, J. Marcoux 2,5, D. Juncker 2,3,4
1 Imperial College London School of Medicine,Ìý2 Dpt. Neurology and Neurosurgery, Montreal Neurological Institute,Ìý3 Dpt. Biomedical Engineering,Ìý4 Â鶹AV and Génome Québec Innovation Center,Ìý5 Montreal General Hospital, Â鶹AV Health Center Research InstituteÌý
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References
1. Ìý Ìý Hillman J, Aneman O, Anderson C, Sjögren F, Säberg C, MellergÃ¥rd P. A microdialysis technique for routine measurement of macromolecules in the injured human brain. Neurosurgery. 2005;56:1264-70.
2. Ìý Ìý Helmy A, Carpenter KL, Skepper JN, Kirkpatrick PJ, Pickard JD, Hutchinson PJ. Microdialysis of cytokines: methodological considerations, scanning electron microscopy, and determination of relative recovery. J Neurotrauma. 2009 Apr;26:549-61.Ìý
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