Expression analysis following argon treatment in an in vivomodel of transient middle cerebral artery occlusion in rats
© Fahlenkamp et al.; licensee BioMed Central Ltd. 2014
Received: 23 April 2014
Accepted: 29 May 2014
Published: 6 June 2014
Argon treatment following experimental neurotrauma has been found neuroprotective in an array of in vivo and in vitro models. The inherent cellular and molecular mechanisms are still unknown. We seeked to shed light on these processes by examinig the cellular distribution and the expression of inflammatory markers and growth factors in argon treated brain tissue.
Male adult Sprague-Dawley rats were randomly assigned to one of the study groups: sham surgery + placebo, sham surgery + argon, tMCAO + placebo, and tMCAO + argon. Animals underwent 2 h-transient middle cerebral artery occlusion (tMCAO) using the endoluminal thread model or sham surgery without tMCAO. After the first hour of tMCAO or sham surgery a 1 h inhalative argon (50% argon/50% O2) or placebo (50% N2/50% O2) treatment was performed. Brains were removed and evaluated after 24 h. RealTime-PCR was performed from biopsies of the penumbra and contralateral corresponding regions. Paraffin sections were immunostained with antibodies against GFAP, NeuN, and Iba1. Cell counts of astrocytes, neurons and microglia in different cortical regions were performed in a double-blinded manner.
Fifteen animals per tMCAO group and twelve sham + placebo respectively eleven sham + argon animals completed the interventional procedure. We identified several genes (IL-1β, IL-6, iNOS, TGF-β, and NGF) whose transcription was elevated 24 h after the study intervention, and whose expression levels significantly differed between argon treatment and placebo following tMCAO. Except for the core region of ischemia, cell numbers were comparable between different treatment groups.
In our study, we found an elevated expression of several inflammatory markers and growth factors following tMCAO + argon compared to tMCAO + placebo. Although conflicting the previously described neuroprotective effects of argon following experimental ischemia, these findings might still be associated with each other. Further studies will have to evaluate their relevance and potential relationship.
Cerebral ischemia and its sequelae is one of the leading causes of death and long-term disability worldwide [1, 2]. In contrast to its common incidence, therapeutic options are limited. Due to the high vulnerability of neuronal tissue to oxygen deprivation, recanalization procedures have to take place in a limited time slot of 3 h and up to 4.5 h in special cases . The area of greatest interest is the so called penumbra: the transition zone in between ischemic core and healthy tissue, where neurons are in a state of hibernation that can be restituted ad integrum with optimum treatment . Numerous experimental therapeutic approaches have focused on protection and salvation of neurons in this area. While most of these therapeutics showed promising results in experimental studies, none to negative effects were seen in clinical trials . Until today, and with limited recommendation, only therapeutic hypothermia has found its way into cerebral ischemia treatment guidelines [1, 2].
The noble gas argon has recently come into focus as a potential adjunctive neuroprotective agent: protective effects of noble gases have been widely shown in different experimental models [4–6], and argon seems to have similar beneficial effects on injured tissue [7–10]. Moreover, unlike xenon, it lacks sedative side effects under normobaric conditions , and is cheaper due to its higher fraction in normal atmosphere. These features plus expectedly little disadvantageous side effects in humans make argon a promising candidate. However, while neuroprotective effects of argon have already been demonstrated, data on its cellular actions are limited: In vitro, argon activated a cellular enzyme, and it only slightly interfered with the inflammatory microglial response following stimulation with bacterial lipopolysaccharide . In vivo, an elevated expression of an anti-apoptotic protein was found elevated following argon treatment in neonatal asphyxia . Argon’s protective action was not exerted via NMDA-antagonism by interaction with glycine in a model of traumatic brain injury . The mechanisms for argon’s neuroprotective actions in cerebral ischemia are unknown.
The aim of this study was to shed first light on argon’s mechanisms of action in a stroke model. 24 h after transient middle cerebral artery occlusion (tMCAO)  and a delayed additional administration of 50% argon as described previously , we performed a gene expression analysis of inflammatory and growth factors and examined the distribution of vital neurons, microglia and astrocytes in the penumbra.
Study design and animal enrolment
This experimental study was in part designed as a subgroup- and follow-up analysis of the above mentioned efficacy analysis of argon in MCAO , with additional enrolment of animals obtaining the same experimental procedures. Animal research and care procedures were approved by the governmental review board (Landesamt für Natur, Umwelt und Verbraucherschutz, Germany) beforehand. Species-appropriate housing was assured in macrolone cages in a specified pathogen-free environment, with food and water ad libitum and a 12 h-light-dark-cycle.
Transient middle cerebral artery occlusion (tMCAO)
Male Sprague Dawley rats (250-295 g, Harlan Laboratories, Netherlands) were randomly assigned to one of the following groups: a) sham surgery + placebo (Sham N2): n = 12; b) sham surgery + argon treatment (Sham Ar): n = 12; c) tMCAO + placebo (tMCAO N2): n = 15; and d) tMCAO + argon (tMCAO Ar): n = 15. One animal in the Sham Ar group was lost before completion of the 24 h follow-up due to other than intervention-associated reasons.
Tissue preparation for gene expression analysis
V edi = Volume edema-corrected infarct volume, V infarct = Volume infarct, V ipsi = Volume ipsilateral infarcted hemisphere, V contra = Volume contralateral non-infarcted hemisphere.
On the basis of TTC-staining, the penumbra regions of the infarcted brain hemispheres were identified. Total RNA was isolated from the cortical penumbra of the infarcted hemisphere and corresponding contralateral regions from three consecutive coronal sections of each brain (sections 3, 4 and 5) employing NucleoSpin RNA II (MACHEREY-NAGEL, Germany) according to the manufacturers’ instructions. Briefly, brain tissue was homogenized (5000 g, 15 s) with Precellys Keramik-Kit (PEQLAB Biotechnologie, Germany). Cells were further lysated with β-mercaptoethanol, filtrated by centrifugation and washed in 70% ethanol. DNA was digested and RNA purified in several washing steps and eluted in ultra-purified water (Invitrogen, Germany).
Gene expression analysis
Primer sequence, product length and characteristics
Annealing temperature (°C)
Transcript size (base pairs)
Melting point (°C)
Tumor nekrose faktor alpha
Chemokine (C-C) ligand 2
Hypoxia inducible factor 1
Chemokine (C-C) ligand 5
Transforming growth factor β (TGF-β)
Nerve growth factor
Vascular endothelial growth factor α (VEGFα)
2’3’-Cyclic nucleotide 3’ phosphodiesterase (CNP)
Glial fibrillary acid protein
Insulin-like growth factor 1
The primer sequences and further information about the transcripts we analyzed is given in Table 1. We selected the transcripts for analysis as follows: Argon was found to have effects on both glial and neuronal cells, and it only slightly attenuated LPS-induced production of cytokines IL-1β, TNFα and IL-6 in a microgial cell line . HIF-1α was found to be induced in xenon but not in argon renal cell protection . Since knowledge about argon effects is in general very limited, additional factors that are released by neuronal or glial or invading immune cells and are furthermore associated with damage and/or repair following neurotrauma were chosen for analysis.
In deep anesthesia, rats were transcardially perfused with 2% (w/v) paraformaldehyde (Roth, Germany) containing 15% (v/v) saturated picric acid at pH 7.4 (AppliedChem, Germany). Brains were removed, kept in the same fixative overnight and paraffin-embedded the next day (Merck, Germany). Coronary sections (slices 3 and 5) of 5 μm thickness were prepared, mounted on glass slides and rehydrated using standard protocols. To verify infarction (MCAO) or integrity (Sham), sections were stained with hematoxylin/eosin (HE) using a standard protocol (Exemplary pictures are presented in Figure 1B). For IHC, sections were incubated with blocking serum for 1 h at room temperature, following exposure to primary antibodies overnight (anti-NeuN (1:500, Abcam, Cambridge, UK), anti-GFAP (1:1000, Abcam, Cambridge, UK), and anti-ionized calcium-binding adaptor molecule 1 (Iba1, 1:250, Wako, Osaka, Japan). After several washing steps, sections were incubated with biotin-conjugated secondary antibody for 1 h, and subsequently with a biotin–avidin–enzyme complex (Vectastain ABC kit, Vector Laboratories, Burlingame, USA). Antibody binding was visualized by substrate incubation (AEC Substrate kit, Invitrogen, Camarillo, USA). To quantify vital cells, standard light microscopy with a 20× objective lens was performed and images were taken of three defined regions (healthy cortex, cortex penumbra and ischemic core, see Figure 1B) in the infarcted hemisphere and of corresponding contralateral regions with a standard digital camera. Vital neurons (NeuN positive cells with a vital large light caryon without shrinked/dense apperance), astrocytes (GFAP-positive cells with minimum three branches) and microglia (Iba1-positive cells with minimum two rami) were analyzed with the help of a cell count software (ImageJ, http://rsbweb.nih.gov/ij/) by two independent examiners blinded to the treatment.
Parametric data were evaluated by oneway ANOVA, followed by post-hoc Newman-Keuls-testing for statistical significance. GraphPad PRISM (GraphPad Software Inc., La Jolla, California, USA) was used to calculate the statistics and generate the figures. All values are given as means ± SEM. A p-value ≤ 0.05 was considered statistically significant.
Expression of inflammatory genes
Expression of growth factors
Distribution of neurons, microglia, and astrocytes
Microglia counts were comparable in healthy contralateral hemispheres in all study groups (data not shown). Microglia numbers were not significantly different among treatment groups neither in the ischemic nor in the penumbra or healthy region (Figure 5B). Example stainings are shown in Figure 6B.
Astrocyte numbers were not significantly different among treatment groups neither in the ischemic (Figure 5C) nor in the contralateral hemispheres (data not shown). Example stainings are shown in Figure 6C.
Recent experimental studies were able to demonstrate neuroprotective properties of argon in different ischemic and traumatic settings [6–10, 19]. However, cellular and molecular processes leading to this neuroprotection are largely unknown. With this study, we intended to start elucidating protective mechanisms of argon treatment in a model of transient focal cerebral ischemia in rats. We identified several genes whose transcription was elevated 24 h after intervention, and whose expression levels significantly differed between argon treatment and placebo following tMCAO. Apart from the ischemic core in tMCAO + placebo animals, numbers of astrocytes, microglia and neurons were not significantly different in the study groups.
Following tMCAO + argon, several inflammatory markers showed significantly higher expression levels in the argon group at 24 h post interventionem. The cytokines IL-1β and IL-6 are regularly secreted by neurons and glia in response to an ischemic stimulus and are involved in macrophage attraction and binding . Especially IL-1β is actively produced and released in stroke in tattered neurons and activated microglia by activation of inflammasomes and apoptotic pathways in the hypoxic brain. These differences in gene expression seem contradictory to the neuroprotective effects exerted by argon in tMCAO in rats . The cytokines have mostly been found to aggravate tissue damage in ischemia [20–22]. IL-6 has also been attributed a protective role [23, 24]. About both the underlying pathomechanisms and the prospects of these unexpected results we may only speculate. Direct as well as subordinate effects of argon on the injured tissue might be underlying these findings. Of note, expression kinetics of these cytokines showed an immediate up-regulation approximately one to two hours after brain injury induction with peak levels after 6 to 12 h [25, 26]. Since in our study mRNA levels were measured with a delay of 24 h after reperfusion, it might be thinkable that besides a prolonged upregulation of these cytokines, argon might induce a delay of mRNA-expression which might effect a shift of peak expression to a later time point. At 24 h post-ischemia the elevated expression of iNOS, whose role in ischemic brain damage and repair is still discussed [27, 28], might also be due to altered cytokine kinetics. Another important point to consider is that the timing of sampling might contribute to these confusing results. Only having the 24-hour snap shot might not be sufficient to draw mechanistic conclusions. Both earlier (first 6-to-12 hours of post-ischemia) and later (48 hours post-ischemia) sampling will have to be evaluated in further studies to examine these seemingly controversial results and their link to tissue protection.
Growth factors are involved in initiation of repair mechanisms to organize tissue damage and initiate cell migration and differentiation. TGF-β-expression was found to be elevated 24 h after tMCAO + argon while it was not affected by tMCAO + placebo. TGF-β is associated with profound neuroprotective activities [29, 30]. NGF, which was also induced by argon treatment in our study, has been shown to exert neuroprotective properties partly via an antiapoptotic mechanism . VEGFα plays a role in regulation of cerebral blood flow and angiogenesis and thereby is proposed to act as a neuroprotectant . The fact that these neuroprotective factors were increased following argon therapy might contribute to its beneficial effects .
Tissue protection by xenon preconditioning and treatment has been associated with an up-regulation of HIF-1α in several in vivo and in vitro models of ischemia [18, 33, 34]. Argon preconditioning did not induce HIF-1α in renal ischemia in vitro, but also lacked protective effects in this experiment . In our study, we did not find an upregulated expression of HIF-1α following argon treatment of tMCAO induced cerebral ischemia despite of it’s neuroprotective effects . Whether the missing induction of HIF-1α is specific to argon, or due to other reasons (timing of treatment, examination time) will have to be determined elsewhere.
Still, some open questions remain: There was no statistically significant difference in neuron numbers between tMCAO + argon and tMCAO + placebo in the penumbra, although behavioural scoring and cortical infarct volumes in tMCAO + argon animals had been improved compared to placebo treatment . A higher number of animals might be required to establish such a correlation. Alternatively, the reason for this finding could be that the behavioural scoring does not reflect the neuronal situation in the penumbra. Furthermore, we found several notable changes in the expression of the above named genes, but their exact cellular source remains to be identified. Additional studies will have to address these questions.
In our study, we found controversial results. Besides elevated expressions of several inflammatory cytokines (IL-1β, IL-6, iNOS) we found an increased expression of neuroprotective growth factors (TGFβ, NGF, VEGF) following tMCAO + argon compared to tMCAO + placebo. These findings might be associated with the previously described neuroprotective effects of argon following experimental neurotrauma. Further studies will have to evaluate the relevance of these findings.
We thank the team at the Department of Animal Research, University Hospital Aachen for expert laboratory advice, assistance and help. We further thank J. Hoffmann and R. Debarry for their excellent technical assistance.
This research was conducted with funding by the Rotationsprogramm (YMR, AVF) and the START programm (AVF), both Medical Faculty, RWTH Aachen.
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