Treatment of brain tumours with electroporation and bleomycin in an in vivo rat model

The purpose of the study was to examine the possibilities of prolonging the life of rats with brain tumours using electroporation only while conducting impedance scans to evaluate the rate of electroporation. During the experiments, the treated rats in the first batch had tumors grow for 14 days and were then given electroporation with 8 + 8 exponential pulses at 800 v/cm, and 15 micro-F. Three of the animals given electroporation and bleomycin and one of the animals given electroporation alone died within three days of treatment. The reason for this may be oedema and increased intracranial pressure due to the large amount of energy delivered to the brain. In the second batch of rats given electroporation with 8 + 8 exponential pulses at 800 V/cm but with the lower capacitance setting of 4.5 micro-F, none of the treated rats died within a week after treatment. Among these rats, the tumors had also grown for only 10 days, thus further decreasing the risk of critical increases of the intracranial pressure directly after treatment. Interpretation of the impedance scans made before and after is made difficult due to factors such as possible short-circuiting of electrodes due to bleeding in the operating area. Not counting the rats in the first batch that died within three days of treatment, the rats treated with electroporation had a significantly prolonged life compared to controls. These results combined with results of a prior study of treatment of brain tumors in rats with electroporation in combination with Bleomycin suggest that this form of treatment may prolong the life of patients with brain tumours

Blood-brain barrier permeability in rats exposed to electromagnetic fields used in wireless communication

iological effects of radio frequency electromagnetic fields (EMF) on the blood-brain barrier (BBB) have been studied in Fischer 344 rats of both sexes. The rats were not anaesthetised during the exposure. All animals were sacrificed by perfusion–fixation of the brains under chloralhydrate anaesthesia after the exposure. The brains were perfused with saline for 3–4 minutes, and thereafter perfusion fixed with 4% formaldehyde for 5–6 minutes. Whole coronal sections of the brains were dehydrated and embedded in paraffin and sectioned at 5 m. Albumin and fibrinogen were demonstrated immunohistochemically and classified as normal versus pathological leakage. In the present investigation we exposed male and female Fischer 344 rats in a Transverse Electromagnetic Transmission line chamber to microwaves of 915 MHz as continuous wave (CW) and pulse-modulated with different pulse power and at various time intervals. The CW-pulse power varied from 0.001 W to 10 W and the exposure time from 2 min to 960 min. In each experiment we exposed 4–6 rats with 2–4 controls randomly placed in excited and non-excited TEM-cells respectively. We have in total investigated 630 exposed rats at various modulation frequencies and 372 controls. The frequency of pathological rats is significantly increased (p < 0:0001) from 62=372 (ratio: 0:170:02) for control rats to 244=630 (ratio: 0:390:03) in all exposed rats. Grouping the exposed animals according to the level of specific absorbed energy (J/kg) give significant difference in all levels above 1.5 J/kg. The exposure was 915 MHz microwaves either pulse modulated (PW) at 217 Hz with 0.57 ms pulse width, at 50 Hz with 6.6 ms pulse width or continuous wave (CW). The frequency of pathological rats (0:17) among controls in the various groups is not significantly different. The frequency of pathological rats was 170=481 (0:350:03) among rats exposed to pulse modulated (PW) and 74=149 (0:500:07) among rats exposed to continuous wave exposure (CW). These results are both highly significantly different to their corresponding controls (p < 0:0001) and the frequency of pathological rats after exposure to pulsed radiation (PW) is significantly less (p < 0:002) than after exposure to continuous radiation (CW)

Brain tumour growth in rats exposed to electromagnetic fields used in wireless cellular communication

In 1996 there was no convincing laboratory evidence that EMFs used in wireless communication could cause tumour promotion at non-thermal exposure levels. Therefore we then performed a study of the effects from exposure to such electromagnetic fields in the rat brain glioma model we were using in our research for brain tumour therapy. By stereotaxic technique rat glioma cells (RG2 or N32) were injected into the head of the right caudate nucleus in 154 pairs of Fischer 344 rats in both exposed and matched controls. Starting on day 5 after inoculation, the animals were exposed for 7 hours a day, 5 days a week during 2 - 3 weeks. Rats of both sexes were exposed to electromagnetic fields in the microwaves frequency range 915 MHz both as continuous waves (1 W), and as pulse-modulated at 4, 8, 16 and 217 Hz in 0.57 ms long pulses and 50 Hz in 6.67 ms pulses, all with a maximum power amplitude of 2 W per pulse. The animals were kept un-anaesthetized in well-ventilated TEM cells during 7 hours a day for 5 days a week for 2-3 weeks. Their matched controls were kept in identical TEM cells without EMF exposure. At the end of the exposure period the rat brains were examined histopathologically. The tumour size was measured with a calliper and the volume estimated as an ellipsoid. Our study of the 154 matched pairs of rats did not show any significant difference in tumour volume between animals exposed to 915 MHz microwaves, and those not exposed. Thus our results did not support that daily exposure to EMF promotes tumour growth when given from the fifth day after the start of tumour growth in the rat brain until the sacrifice of the animal 16 days later. In the present review our results published 1997 have been re-evaluated in terms of SAR dependence of tumour volume observed ratio (exposed / control). We thus surprisingly found that the shape of tumour volume-OR versus SAR response was of bath-tube pattern, similar to that found in our parallel studies of albumin leakage through the blood-brain barrier. Since the SAR varies between most other animal studies reviewed and human epidemiological studies this SAR dependence might explain the controversy in rendering the results

STUDIES OF EFFECTS OF GSM-900 MICROWAVE EXPOSURE ON DNA ”MICRONUCLEUS” FORMATION IN MICE

Possible genotoxic effects of microwave exposure from GSM-900 mobile telephones have investigated with in vivo micronucleus assay of mouse erythrocytes from CBA mice and GFAP knockout mice. No significant change in the frequency of erythrocytes with micronuclei neither in the young (polychromatic PCE) or mature (normichromatic NCE) erythrocytes. There is, however, a tendency but not significant to increased MPCE in female mice after 35 days of exposure. There is a marked tendency to lower PCE-fraction in the exposed groups. When male and female is studied separately there is no significant difference. However, if the values are normalised to eliminate the sex-difference there is a significant lower fraction in the exposed mice. Another observation is lower weight of the exposed male. If normalised data for both sexes are pooled there is an almost significant difference (95% level) in weight. We found a less pronounced difference in the CBE mice than in the GFAP experiment. Thus genotype might play a role in microwave exposure. Differences in exposure time and number of controls in GFAP and CBA experiment might influence the results. We observe a moderate decrease of formation of new erythrocytes in the exposed animals. This might fit the tendency of lower weight in the exposed animals and might indicate a general decreased cell-proliferation in the exposed animals

S-100 protein levels in the blood of Fischer rats exposed to 915 MHz CW-microwaves and magnetic fields.

EnglishPersson, B. R. R., Nittby Redenbrant, H., Malmgren, L., Brun, A., and Salford, L. G. (2020). S-100 protein levels in the blood of Fischer rats exposed to 915 MHz CW-microwaves and magnetic fields. Acta Scientiarum Lundensia, Vol. 2020-005, pp. 1-9, ISSN 1651-5013Abstrakt.Syftet var att studera nivån av S-100 i blodprover som tagits från Fischer-344-råttor efter exponering för 915 MHz CW-mikrovågor och ELF-magnetfält i TEM-celler. Magnetfältsexponering ägde rum med TEM-cellen i ett Helmholtz-spolarrangemang med antingen 50 Hz sinusformat magnetfält på 5 µT eller inkoherent magnetfältbrus IMF med en maximal amplitud på 50 µT.Det verkar inte finnas någon signifikant förändring i S-100-koncentrationen i blod hos råttor som exponerats under 6 timmar för höga nivåer (4W) av kontinuerlig våg (CW) 915MHz mikrovågor, 50Hz sinusformade magnetfält (5µT) och osammanhängande magnetfält (IMF) vid 50µT. Däremot indikerar resultaten av den kombinerade exponeringen CW + IMF en minskad koncentration av S100 i blod.Minskningen överensstämmer med resultaten av en undersökning under 2010 att de extremt lågfrekventa (ELF) magnetfälten från fläktmotorn (50Hz AC, 0,3-1,5 T) minskade det förväntade BBB-läckaget av albumin på grund av mikrovågsexponering .Resultaten från andra studier indikerar också att bioeffekter orsakade av exponering för mikrovågor reduceras genom överläggning med extremt lågfrekventa magnetfält ELF. Det är därför av yttersta vikt när man undersöker bioeffekter från mikrovågor för att också kontrollera nivåer och frekvenser av lågfrekventa magnetfält i omgivningen, vilket kan vara en av anledningarna till den breda spridningen i de rapporterade resultaten

Non-thermal" Effects on the Blood-Brain Barrier in Fischer rats by exposure to microwaves

Effect of 915 MHz electromagnetic fields (EMF) on the blood brain-barrier (BBB) permeability has been studied in Fischer 344 rats of both sexes. Male and female Fischer 344 rats were exposed in a Transverse Electromagnetic Transmission line chamber to microwaves of 915 MHz as continuous wave (CW) and pulse-modulated with different pulse power and at various time intervals. The CW-pulse power varied from 0.001W to 10 W and the exposure time from 2 min. to 960 min. In each experiment we randomly placed 4 rats in excited and 4 control rats in non-excited TEM-cells respectively. The rats were not anaesthetised during the exposure. The rats were exposed to 915 MHz microwaves, either continuous wave (CW) or pulse modulated at 4,8,16 or 217 Hz with 0.57 ms pulse width, or pulse modulated at 50 Hz with 6.6 ms pulse width as well as from a real GSM-900 telephone. All animals were sacrificed by perfusion-fixation of the brains under chloralhydrate anaesthesia after the exposure. The brains were then perfused, first with saline for 3-4 minutes, and then with 4% formaldehyde for 5-6 minutes. Whole coronal sections of the brains were dehydrated and embedded in paraffin and sectioned at 5 μm. The degree of albumin leakage was demonstrated immune-histo-chemically and classified in order of increased number of albumin extravasations by a rank number: 0 - 0.5 - 1.0 - 1.5 - 2 - 3. Pathological albumin leakage was judged as albumin extravasations equal to or larger than 1. The frequency of pathological rats in all control groups was about 17%. Among rats exposed to pulse modulated microwaves the ratio of pathological rats was 170/481(0.35±0.03) and among rats exposed to continuous wave exposure (CW) it was 74/149 (0.50±0.07). These results are both highly significantly different to their corresponding controls (p<0.0001). The rats were exposed to SAR various values: 0.2; 2; (20-40); (100-500); (1000-3000) mW/kg. In the 217 Hz modulated group (GSM simulated) we found the most increased ratio of albumin extravasations OR= 4 at 0.2 mW/kg. But no significant increased ratio at SAR 2000 mW/kg. The response curve of OR versus log(SAR) had the shape of a bathtub, with a minimum at a100 mW/kg. A similar curve was recorded for OR versus Specific Absorbed Energy (SAE Joule / kg) with a minimum at 100 J/kg. Similar response curves were recorded for the various modulation frequencies 4; 8; 16; 50 Hz. We found no pronounced difference between the various modulation frequencies other than the effect of CW exposure seems to be more effective than pulse modulated exposure in opening the BBB at high SAR values 100-2000 mW/kg. Conclusion: The opening of the BBB is most effective at SAR values in the range of 0.1-0.5 mW.kg-1 and less effective in the range of 50-500 mW.kg-1. In this low SAR range thermal effects are unlikely. Thus there seems to be a non-thermal mechanism involved triggering the opening of the BBB

Immunization with syngeneic interferon-gamma (IFN-g) secreting tumour cells enhance the Therapeutic effect and Abscopal effect from combined treatment of subcutaneously implanted contra-lateral N29 tumours on Fischer rats with Pulsed electric fields (PEF) and 60Co-gamma radiation.

The aim of the present study is to study the Abscopal regression of subcutaneously implanted N29 rat glioma after immunization with syngeneic IFNg secreting cells and treatment of contra-lateral tumours with pulsed electric fields (PEF) and/or radiation therapy (RT). The study was performed on rats of the Fischer-344 strain with rat glioma N29 tumours implanted subcutaneously on the flank or on both the right treated hind leg and the left untreated hind leg. Once weekly for three weeks, the animals were given intra-peritoneal injections of irradiated, modified N29 tumour cells, secreting interferon-gamma (IFN-g). PEF was given with 16 exponentially decaying pulses at a maximum electric fields strength of 1400 V/cm and t1/e= 1 ms. RT was given with 60Co gamma radiation at daily fractions of 5 Gy, to a total absorbed dose of 20 Gy. The animals were arranged into controls and groups of various treatments: PEF, RT, PEF+RT and immunization (IFNg). Fitting the data obtained from consecutive measurements of tumour volume (TV) of each individual tumour to an exponential model TV = TV0*exp[TGR*t] estimated the tumours growth rate (TGR %per day) after the day of treatment (t = 0). TGR of the right-lateral treated tumour was significantly decreased for independent treatments with PEF and RT and with the combined treatment PEF+RT. With immunization (IFNg) alone and in combination with PEF there was, however, no significant decrease of the TGR of the right-lateral tumours. But in the combination of immunization with RT or PEF+RT there was a highly significant decrease of the TGR values. The Abscopal effect was evaluated by comparing the growth rate of the untreated contra lateral tumours with the treated tumours. TGR of the left-lateral untreated tumour in the groups with independent treatment of right-lateral tumours with PEF, was not significantly reduced. But the TGR values are significantly reduced in the group of rats treated with RT and the combination PEF + RT. With IFNg alone and in combinations with PEF or RT there was no significant decrease of the TGR in the left lateral tumours. But in the combination of IFNg with PEF+RT there was a highly significant decrease of the TGR values in the left lateral tumours. The specific therapeutic effect (STE = 1 - TGRExposed/ TGRCtrl ) after treatments with PEF was 0.30±0.01 and after RT 0.46±0.04 and after the combination PEF+RT 0.36+/- 0.08. After immunization with IFNg secreting tumour cells the STE 0.09+/- 0.07 is not significantly different from zero. Also for the combination of immunization and PEF the STE value of 0.07+/- 0.07 is not significantly different from zero. In the combination of immunization with RT the STE value was 0.32+/- 0.01 that is significantly different from zero and only slightly lower than for RT alone. The STE of the combination of immunization with (PEF+RT) resulted in an unexpectedly high STE value of 0.70+/- 0.08 that is highly significantly different from zero (p < 0.0001). The specific Abscopal effect (SAE = 1 - TGRUn-Exposed/ TGRCtrl ) of the contra lateral unexposed tumours in rats treated with PEF or RT are both significantly different from zero. For RT the average SAE value is 0.33+/- 0.04 and for PEF it is 0.11+/- 0.05. The SAE value for the combined treatment with PEF + RT is 0.26+/- 0.02 that is about the same as for RT alone. For immunization with IFNg secreting tumour cells only and IFNg +PEF the SAE values were not significantly different from zero. But IFNg combined with RT result in a SAE value of 0.18±0.12 and the combination of IFNg with PEF+RT results in an improved abscopal effect with the SAE value of 0.33+/- 0.06. After combined treatment with PEF + RT the average of the therapeutic enhancement ratio (TER = STEExperimental / STEIndependent) is 0.47 +/- 0.12 and the abscopal enhancement ratio (AER = SAEExperimental / SAEIndependent) is 0.61 +/- 0.1 respectively. With all three treatment modalities combined IFNg + PEF + RT and all combinations of independent treatments with PEF, RT or IFNg are considered, the average of the TER is 1.20+/- 0.15 and AER is 1.22+/- 0.20. This might indicate that there is a synergism on the tumours on both sides by combining PEF, RT and immunization with IFNg secreting cells. These results were first presented Nov 21-24, 2002, as Poster at Society of Neuro-Oncology (SNO) Annual Meeting, San Diego, USA (Persson et al 2002)

Efficient internalization into low-passage glioma cell lines using adenoviruses other than type 5: an approach for improvement of gene delivery to brain tumours

There is a need for improvement of the commonly used adenovirus vectors based on serotype 5. This study was performed on three adenovirus serotypes with a CAR-binding motif (Ad4p, Ad5p and Ad17p) and three non-CAR-binding serotypes (Ad11p, Ad16p and Ad21p). The capacity of these alternative adenovirus vector candidates to deliver DNA into low-passage glioma cell lines from seven different donors was evaluated. The non-CAR-binding serotype Ad16p was the most efficient serotype with regard to import of its DNA, as well as initiation of hexon protein expression. Ad16p established hexon expression in 60–80 % of the cell population in gliomas from all donors tested. The other non-CAR-binding serotypes, Ad11p and Ad21p, showed hexon expression in 25–60 and 40–80 % of cells, respectively. The corresponding figure for the best CAR-binding serotype, Ad5p, was only 25–65 %, indicating greater variability between cells from different donors than serotype Ad16p had. The other CAR-binding serotypes, Ad4p and Ad17p, were refractory to some of the gliomas, giving a maximum of only 45 and 40 % hexon expression, respectively, in the most permissive cells. Interestingly, the transduction capacity of the CAR-binding serotypes was not correlated to the level of CAR expression on the cells

Nerve cell damage in mammalian brain after exposure to microwaves from GSM mobile phones.

The possible risks of radio-frequency electromagnetic fields for the human body is a growing concern for our society. We have previously shown that weak pulsed microwaves give rise to a significant leakage of albumin through the blood-brain barrier. In this study we investigated whether a pathologic leakage across the blood-brain barrier might be combined with damage to the neurons. Three groups each of eight rats were exposed for 2 hr to Global System for Mobile Communications (GSM) mobile phone electromagnetic fields of different strengths. We found highly significant (p< 0.002) evidence for neuronal damage in the cortex, hippocampus, and basal ganglia in the brains of exposed rats

Zebularine induces long-term survival of pancreatic islet allotransplants in streptozotocin treated diabetic rats.

Coping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed