Photobiomodulation Activates Microglia/Astrocytes and Relieves Neuropathic Pain in Inferior Alveolar Nerve Injury.

Objective: This study aimed to determine microglial/astrocyte changes and their associated analgesic effect in inferior alveolar nerve injury (IANI) model rats treated with photobiomodulation therapy (PBMT) using a 940-nm diode laser. Background: Very few basic studies have investigated microglial/astrocyte dynamics following PBMT aimed at relieving neuropathic pain caused by IANI. Methods: Rats were divided into an IANI-PBM group, IANI+PBM group, and sham+PBM group. Observations were made on the day before IANI or the sham operation and on postoperative days 3, 5, 7, 14, and 28. PBMT was delivered for 7 consecutive days, with an energy density of 8 J/cm2. Behavioral analysis was performed to determine pain thresholds, and immunohistological staining was performed for the microglia marker Iba1 and astrocyte marker glial fibrillary acidic protein, which are observed in the spinal trigeminal nucleus. Results: Behavioral analysis showed that the pain threshold returned to the preoperative level on postoperative day 14 in the IANI+PBM group, but decreased starting from postoperative day 1 and did not improve thereafter in the IANI-PBM group (p ≤ 0.001). Immunological analysis showed that microglial and astrocyte cell counts were similar in the IANI+PBM group and IANI-PBM group shortly after IANI (day 3), but the expression area was larger (p ≤ 0.001) and hypertrophy of microglia and astrocyte cell bodies and end-feet extension (i.e., indicators of activation) were more prominent in the IANI+PBM group. Conclusions: PBMT after IANI prevented hyperalgesia and allodynia by promoting glial cell activation shortly after injury.

Time-dependent photobiomodulation management of neuropathic pain induced by spinal cord injury in male rats.

Neuropathic pain (NP) following spinal cord injury (SCI) often lasts for a long time and causes a range of problems that reduce the quality of life. Current treatments are not generally effective; however, photobiomodulation therapy (PBMT) has made some progress in this area. Due to the novelty of this treatment, standard therapeutic protocols have not yet been agreed upon. In the present study, we compare the analgesic effect of two PBMT protocols (2 and 4 weeks of radiation). A total of thirty-two adult male Wistar rats were divided into four groups: control, SCI, 2 W PBMT, and 4 W PBMT. SCI was induced by an aneurism clip and PBMT used a 660-nm, initiated 30 min post-SCI, and continued daily for 2 or 4 weeks. Functional recovery, hyperalgesia, and allodynia were measured weekly. At the end of the study, the Gad65, interleukin 1-alpha (IL1α), interleukin 10 (IL10), IL4, and purinergic receptor (P2xR and P2yR) expressions were measured. Data were analyzed by Prism6. The results showed PBM irradiation for 2 and 4 weeks had the same effects in improving hyperalgesia. In the case of allodynia and functional recovery, 4 W PBMT was more effective (p<0.01). 4 W PBMT increased the Gad65 expression (p <0.001) and reduced P2Y4R (p <0.05) compared to SCI animals. The effects of 2 and 4 W PBMT were the same for IL1α, IL10, and P2X3 receptors. 4 W PBMT was more effective in reducing the complications of SCI such as pain and disability. PBMT therapy is an effective method aimed at immune system function modulation to reduce NP and motor dysfunction.

Central involvement of 5-HT1A receptors in antinociception induced by photobiomodulation in animal model of neuropathic pain.

This study aimed to investigate the central involvement of 5-HT1A receptors in the nociceptive behavior of mice submitted to the chronic constriction injury (CCI) of sciatic nerve and the subsequent application of photobiomodulation (PBM). Male mice (Swiss-albino) were submitted to CCI and subsequently received an infusion of WAY100635 (5-HT1A receptor antagonist) or intracerebroventricular saline (ICV), followed by infrared laser irradiation (808 nm), in continuous mode, with the power of 100 mW and a dose of 0 J/cm2 (control group) or 50 J/cm2. The thermal hyperalgesia was evaluated by hot plate test, while mechanical allodynia was evaluated by von Frey filaments. After CCI, animals showed a reduction in the nociceptive threshold (p<0.001) when compared to the sham group. In von Frey test, the CCI + saline + PBM 50 J/cm2 group showed an increase in nociceptive threshold (p<0.001) in all measurement moments in comparison with groups CCI + SALINE + PBM 0 J/cm2, CCI + WAY100635 + PBM 50 J/cm2, and CCI + WAY100635 + PBM 0 J/cm2. Similarly, in hot plate test, CCI + SALINE + PBM 50 J/cm2 group showed an increase in nociceptive threshold after application of PBM at 120 and 180 min. Because of the results found, it can be suggested the involvement of 5-HT1A receptors in the central nervous system, since WAY100635 was able to reverse the antinociceptive effect provided by PBM in animals submitted to CCI.

ET-B receptors involvement in peripheral opioid analgesia induced by light-emitting diode photobiomodulation in male and female mice.

Currently, photobiomodulation therapy (PBMT) is gaining space in the scientific and clinical environment. To help elucidate the importance of irradiance, this study evaluated the effect of two different PBMT irradiances (3.5 and 90 mW/cm2), given a fixed wavelength of 630 nm and a dose of 2 J/cm2, on mechanical hyperalgesia following Complete Freund's Adjuvant (CFA) intraplantar (i.pl.) injection in mice. Additionally, we investigated the role of peripheral opioid and endothelin-B receptors (ETB-R), as well as sex differences in treatment outcome. Different groups of male or female mice were evaluated 6 and 96 h after CFA. Mechanical hyperalgesia was evaluated 30 min after treatments. Naloxone or Bq-788 administration, fifteen minutes before PBMT or Sarafotoxin S6c, helped determine the involvement of peripheral opioid and ETB-Rs on PBMT. Lastly, ETB-Rs skin immunocontent in both sexes was quantified after PBMT consecutive daily treatments. PBMT at an irradiance of 90 mW/cm2, was more effective than 3.5 mW/cm2. Bq-788 and naloxone administration prevented the effects of PBMT and SRTX S6c; however, PBMT did not influence peripheral ETB-Rs immunocontent. The results suggest that irradiance influences PMBT effect; and that activation of ETB-R play a role in peripheral PBMT opioid induced analgesia. Lastly, PMBT effects do not appear to be sex-dependent.

Laser-photobiomodulation on experimental cancer pain model in Walker Tumor-256.

CONTEXT: Cancer Pain is considered a common and significant clinical problem in malignant neoplasms, comprising 20% to 50% of all patients with tumor progression. Laser photobiomodulation (L-PBM) has been used in a multitude of pain events, ranging from acute trauma to chronic articular. However, L-PBM has never been tested in cancer pain. OBJECTIVES: Evaluate hyperalgesia, edema, COX-1, COX-2, IL-10, and Bdkrb1 mRNA in low-level laser irradiated Walker-256 tumor-bearing rats. METHODS: Rat hind paw injected with Walker Tumor-256 (W-256) and divided into six groups of 6 rats: G1 (control) - W-256 injected, G2- W-256 + Nimesulide, G3- W-256 + 1 J, G4- W-256 + 3 Jand G5- W256 + 6 J. Laser parameters: λ = 660 nm, 3.57 W/cm2, Ø = 0.028 cm2. Mechanical hyperalgesia was evaluated by Randall-Selitto test. Plethysmography measured edema; mRNA levels of COX-1, COX-2, IL-10, and Bdkrb1were analyzed. RESULTS: It was found that the W-256 + 1 J group showed a decrease in paw edema, a significant reduction in pain threshold. Higher levels of IL-10 and lower levels of COX-2 and Bdkrb1 were observed. CONCLUSION: Results suggest that 1 J L-PBM reduced the expression of COX-2 and Bdkrb1 and increasing IL-10 gene expression, promoting analgesia to close levels to nimesulide.

Modulation of exercise-induced muscular damage and hyperalgesia by different 630 nm doses of light-emitting diode therapy (LEDT) in rats.

We compared the acute effects of different doses of 630 nm light-emitting diode therapy (LEDT) on skeletal muscle inflammation and hyperalgesia in rats submitted to exercise-induced muscle damage (EIMD). Wistar rats were divided into five experimental groups (n = 5-8/group): sedentary control (CON); exercise + passive recovery (PR); and exercise + LEDT (1.2 J/cm2, 1.8 J; 4.2 J/cm2, 6.3 J; 10.0 J/cm2, 15 J). After 100 min of swimming, the rats in the LEDT groups were exposed to phototherapy on the triceps surae muscle. For mechanical hyperalgesia evaluation, paw withdrawal threshold was assessed before and 24 h after swimming. Immediately after hyperalgesia tests, blood samples were collected to analyze creatine kinase (CK) activity and the soleus muscle was removed for histological and tumor necrosis factor (TNF)-α immunohistological analyses. In all LEDT groups, plasma CK activity was reduced to levels similar to those measured in the CON group. Paw withdrawal threshold decreased in the PR group (- 11.9 ± 1.9 g) when compared to the CON group (2.2 ± 1.5 g; p < 0.01) and it was attenuated in the group LEDT 4.2 J/cm2 (- 3.3 ± 2.4 g, p < 0.05). Less leukocyte infiltration and edema and fewer necrotic areas were found in histological sections of soleus muscle in LEDT (4.2 J/cm2) and LEDT (10.0 J/cm2) groups compared to the PR group. Also, LEDT (4.2 J/cm2) and LEDT (10.0 J/cm2) groups showed less immunostaining for TNF-α in macrophages or areas with necrosis of muscle fibers compared to the PR group. LEDT (4.2 J/cm2, 6.3 J)-reduced muscle inflammation and nociception in animals submitted to EIMD.

Light emitting diode (LED) therapy reduces local pathological changes induced by Bothrops asper snake venom.

The therapeutic effect of the Light Emitting Diode (LED) treatment in two wavelengths (635 or 945 nm) was evaluated in the local pathological alterations induced by Bothrops asper snake venom. Mice received irradiation of infrared LED (120 mW, 945 nm) or red LED (110 mW, 635 nm) applied immediately, 1 and 2 h after venom injection. LED treatment reduced edema formation in the plantar region and gastrocnemius muscle and significantly reduced neutrophil migration and hyperalgesia after the venom injection. Also, both infrared LED and red LED treatment significantly reduced myonecrosis, as revealed by muscle CK and plasma CK levels. Histological analysis corroborated the reduction in the extent of venom-induced myonecrosis. In conclusion, our data demonstrates that PBM with LED light in both red and infrared wavelengths, when applied after envenomation in mice, reduces the extent of myotoxicity, edema, inflammatory infiltrate and hyperalgesia, suggesting that photobiomodulation is a potential therapeutic approach that should be further investigated for the treatment of local effects of Bothrops snakebite.

Trigeminal ganglion transcriptome analysis in 2 rat models of medication-overuse headache reveals coherent and widespread induction of pronociceptive gene expression patterns.

We attempted to gather information on the pathogenesis of medication-overuse headache, as well as on the neurochemical mechanisms through which symptomatic medication overuse concurs to headache chronification. Transcriptional profiles were therefore evaluated as an index of the homeostasis of the trigeminovascular system in the trigeminal ganglion of female rats exposed for 1 month to daily oral doses of eletriptan or indomethacin. We report that both drug treatments change trigeminal ganglion gene expression to a similar extend. Of note, qualitative transcriptomic analysis shows that eletriptan and indomethacin prompt nearly identical, increased expression of genes coding for proteins involved in migraine pathogenesis and central pain sensitization such as neuropeptides, their cognate receptors, prostanoid, and nitric oxide-synthesizing enzymes, as well as TRP channels. These genes, however, were not affected in thoracic dorsal root ganglia. Of note, lowering of orofacial nociceptive thresholds, as well as forepaw hyperalgesia occurred in both indomethacin- and eletriptan-treated rats. Our study reveals that chronic rat exposure to 2 acute headache medications with completely different mechanisms of action prompts pain sensitization with highly similar induction of pronociceptive genes selectively within the trigeminal ganglion. Data further our understanding of medication-overuse headache pathogenesis and provide hints for specific mechanism-based treatment options.

Effects of low-level laser therapy on the modulation of tissue temperature and hyperalgesia following a partial Achilles tendon injury in rats.

The aim of the present study was to evaluate the effects of low-level laser therapy (LLLT) on the modulation of tissue temperature and hyperalgesia following a partial injury to the Achilles tendon in rats. Forty-five rats were randomly divided into three groups: a control group, a group treated with LLLT at a dose of 1.4 J (808 nm, 50 mW, 1.4 J), and a group treated with LLLT at a dose of 2.1 J (808 nm, 50 mW, 2.1 J). LLLT was administered to a single point immediately following the partial injury of the Achilles tendon. Tissue temperature and hyperalgesia were evaluated 6, 24, and 48 hours following the injury. Thus, a significant group-versus-time interaction was found for tissue temperature (F = 4.097, p = 0.001) and hyperalgesia (F = 106.605, p < 0.001), with a greater reduction in theses outcomes in the group that received LLLT at a dose of 2.1 J evaluated 48 hours after the injury. Therefore, LLLT at a wavelength of 808 nm and dose of 2.1 J administered immediately following a partial injury to the Achilles tendon led to a reduction in tissue temperature and hyperalgesia at the injury site in rats, especially 48 hours after injury.

Effect of photobiomodulation therapy (808 nm) in the control of neuropathic pain in mice.

Neuropathic pain can be defined as the pain initiated or caused by a primary lesion or dysfunction of the central or peripheral nervous system. Photobiomodulation therapy (PBM) stands out among the physical therapy resources used for analgesia. However, application parameters, especially the energy density, remain controversial in the literature. Therefore, this study aimed to investigate the PBM effect, in different energy densities to control neuropathic pain in mice. Fifty (50) mice were induced to neuropathy by chronic constriction surgery of the sciatic nerve (CCI), treated with PBM (808 nm), and divided into five groups: GP (PBM simulation), GS (sham), GL10, GL20, GL40 (energy density of 10, 20, and 40 J/cm2, respectively). The evaluations were carried out using the hot plate test and Randall and Selitto test, before and after the CCI surgery, every 15 days during the 90 days experiment. ß-Endorphin blood dosage was also tested. For both the hot plate and Randall and Selitto tests, the GL20 and GL40 groups presented reduction of the nociceptive threshold from the 30th day of treatment, the GL10 group only after day 75, and the GP group did not show any improvement throughout the experiment. The ß-endorphin dosage was higher for all groups when compared to the GP group. However, only the GL20 group and GL40 presented a significant increase. This study demonstrates that PBM in higher energy density (20, 40 J/cm2) is more effective in the control of neuropathic pain.

Photobiostimulation reverses allodynia and peripheral nerve damage in streptozotocin-induced type 1 diabetes.

For better evaluation of the efficacy of low-level laser therapy in treating painful diabetic neuropathy and in protecting nerve fiber damage, we conducted a study with type 1 diabetic rats induced by streptozotocin. It is well known that diabetic peripheral neuropathy is the leading cause of pain in those individuals who suffer from diabetes. Despite the efficacy of insulin in controlling glucose level in blood, there is no effective treatment to prevent or reverse neuropathic damage for total pain relief.Male Wistar rats were divided into saline, vehicle, and treatment groups. A single intraperitoneal (i.p.) injection of streptozotocin (STZ) (85 mg/kg) was administered for the induction of diabetes. The von Frey filaments were used to assess nociceptive thresholds (allodynia). Behavioral measurements were accessed 14, 28, 48, and 56 days after STZ administration. Rats were irradiated with GaAs Laser (Gallium Arsenide, Laserpulse, Ibramed Brazil) emitting a wavelength of 904 nm, an output power of 45 mWpk, beam spot size at target 0.13 cm2, a frequency of 9500 Hz, a pulse time 60 ns, and an energy density of 6,23 J/cm2.The application of four sessions of low-level laser therapy was sufficient to reverse allodynia and protect peripheral nerve damage in diabetic rats.The results of this study indicate that low-level laser therapy is feasible to treat painful diabetic condition in rats using this protocol. Although its efficacy in reversing painful stimuli and protecting nerve fibers from damage was demonstrated, this treatment protocol must be further evaluated in biochemical levels to confirm its biological effects.

Low level laser therapy alters satellite glial cell expression and reverses nociceptive behavior in rats with neuropathic pain.

BACKGROUND: Nerve injury often results in persistent or chronic neuropathic pain characterized by spontaneous burning pain accompanied by allodynia and hyperalgesia. Low level laser therapy (LLLT) is a noninvasive method that has proved to be clinically effective in reducing pain sensitivity and consequently in improving the quality of life. Here we examined the effects of LLLT on pain sensitivity induced by chronic constriction injury (CCI) in rats. CCI was performed on adult male rats, subjected thereafter to 10 sessions of LLLT, every other day, and starting 14 days after CCI. Over the treatment period, the animals were evaluated for nociception using behavioral tests, such as allodynia, thermal and mechanical hyperalgesia. Following the sessions, we observed the involvement of satellite glial cells in the dorsal root ganglion (DRG) using immunoblotting and immunofluorescence approaches. In addition we analyzed the expression levels of interleukin 1 (IL-1ß) and fractalkine (FKN) after the same stimulus. RESULTS: LLLT induced an early reduction (starting at the second session; p ≤ 0.001) of the mechanical and thermal hyperalgesia and allodynia in CCI rats, which persisted until the last session. Regarding cellular changes, we observed a decrease of GFAP (50%; p ≤ 0.001) expression after LLLT in the ipsilateral DRG when compared with the naive group. We also observed a significant increase of pro-inflammatory cytokines after CCI, whereas LLLT dramatically inhibited the overexpression of these proteins. CONCLUSIONS: These data provide evidence that LLLT reverses CCI-induced behavioral hypersensitivity, reduces glial cell activation in the DRG and decreases pro-inflammatory cytokines; we suggest that this involvement of glial cells can be one potential mechanism in such an effect.

Expression of mPGES-1 and IP mRNA is reduced by LLLT in both subplantar and brain tissues in the model of peripheral inflammation induced by carrageenan.

The increase in PGE2 production by microsomal PGE synthase-1 (mPGES-1) in CNS contributes to the severity of the inflammatory and pain responses in the model of edema formation and hyperalgesia induced by carrageenan. PGI2, alike to PGE2, plays an important role in the inflammation. Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and the acute inflammatory process. In this work, we studied the effect of LLLT on the expression of both mPGES-1 and IP messenger RNA (mRNA), in either subplantar or total brain tissues obtained from rats submitted to model of edema formation and hyperalgesia induced by carrageenan administration. The test sample consisted of 30 rats divided into five groups: A1 (control-saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw + LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw + LLLT). The animals from groups A3 and A5 were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red laser with wavelengths of 660 nm and dose of 7.5 J/cm(2) was used. Six hours after carrageenan-induced inflammation, mPGES-1 and prostacyclin receptor (IP) mRNA expression were significantly increased both in subplantar and brain tissues. LLLT was able to reduce both mPGES-1 and IP mRNA expression in subplantar and brain tissues. We suggest that LLLT is able to reduce both inflammation and hyperalgesia observed in the model of edema formation and hyperalgesia induced by carrageenan, by a mechanism involving the decrease in the expression of both mPGES-1 and IP.

Effect of low level laser therapy on chronic compression of the dorsal root ganglion.

Dorsal root ganglia (DRG) are vulnerable to physical injury of the intervertebral foramen, and chronic compression of the DRG (CCD) an result in nerve root damage with persistent morbidity. The purpose of this study was to evaluate the effects of low level laser therapy (LLLT) on the DRG in a CCD model and to determine the mechanisms underlying these effects. CCD rats had L-shaped stainless-steel rods inserted into the fourth and fifth lumbar intervertebral foramen, and the rats were then subjected to 0 or 8 J/cm2 LLLT for 8 consecutive days following CCD surgery. Pain and heat stimuli were applied to test for hyperalgesia following CCD. The levels of TNF-α, IL-1ß and growth-associated protein-43 (GAP-43) messenger RNA (mRNA) expression were measured via real-time PCR, and protein expression levels were analyzed through immunohistochemical analyses. Our data indicate that LLLT significantly decreased the tolerable sensitivity to pain and heat stimuli in the CCD groups. The expression levels of the pro-inflammatory cytokines TNF-α and IL-1ß were increased following CCD, and we found that these increases could be reduced by the application of LLLT. Furthermore, the expression of GAP-43 was enhanced by LLLT. In conclusion, LLLT was able to enhance neural regeneration in rats following CCD and improve rat ambulatory behavior. The therapeutic effects of LLLT on the DRG during CCD may be exerted through suppression of the inflammatory response and induction of neuronal repair genes. These results suggest potential clinical applications for LLLT in the treatment of compression-induced neuronal disorders.

Low-level laser therapy (LLLT) reduces the COX-2 mRNA expression in both subplantar and total brain tissues in the model of peripheral inflammation induced by administration of carrageenan.

In the classical model of edema formation and hyperalgesia induced by carrageenan administration in rat paw, the increase in prostaglandin E2 (PGE2) production in the central nervous system (CNS) contributes to the severity of the inflammatory and pain responses. Prostaglandins are generated by the cyclooxygenase (COX). There are two distinct COX isoforms, COX-1 and COX-2. In inflammatory tissues, COX-2 is greatly expressed producing proinflammatory prostaglandins (PGs). Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and acute inflammatory process. Herein we studied the effect of LLLT on both COX-2 and COX-1 messenger RNA (mRNA) expression in either subplantar or brain tissues taken from rats treated with carrageenan. The experiment was designed as follows: A1 (saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw + LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw + LLLT). Animals from the A3 and A5 groups were irradiated at 1 h after carrageenan administration, using a diode laser with an output power of 30 mW and a wavelength of 660 nm. The laser beam covered an area of 0.785 cm(2), resulting in an energy dosage of 7.5 J/cm(2). Both COX-2 and COX-1 mRNAs were measured by RT-PCR. Six hours after carrageenan administration, COX-2 mRNA expression was significantly increased both in the subplantar (2.2-4.1-fold) and total brain (8.65-13.79-fold) tissues. COX-1 mRNA expression was not changed. LLLT (7.5 J/cm(2)) reduced significantly the COX-2 mRNA expression both in the subplantar (~2.5-fold) and brain (4.84-9.67-fold) tissues. The results show that LLLT is able to reduce COX-2 mRNA expression. It is possible that the mechanism of LLLT decreasing hyperalgesia is also related to its effect in reducing the COX-2 expression in the CNS.

Analgesic effect of He-Ne (632.8 nm) low-level laser therapy on acute inflammatory pain.

OBJECTIVE: The aim of this study was to evaluate the analgesic effect of the low level laser therapy (LLLT) with a He-Ne laser on acute inflammatory pain, verifying the contribution of the peripheral opioid receptors and the action of LLLT on the hyperalgesia produced by the release of hyperalgesic mediators of inflammation. BACKGROUND DATA: All analgesic drugs have undesired effects. Because of that, other therapies are being investigated for treatment of the inflammatory pain. Among those, LLLT seems to be very promising. MATERIAL AND METHODS: Male Wistar rats were used. Three complementary experiments were done. (1) The inflammatory reaction was induced by the injection of carrageenin into one of the hind paws. Pain threshold and volume increase of the edema were measured by a pressure gauge and plethysmography, respectively. (2) The involvement of peripheral opioid receptors on the analgesic effect of the laser was evaluated by simultaneous injection of carrageenin and naloxone into one hind paw. (3) Hyperalgesia was induced by injecting PGE2 for the study of the effect of the laser on the sensitization increase of nociceptors. A He-Ne laser (632.8 nm) of 2.5 J/cm2 was used for irradiation. RESULTS: We found that He-Ne stimulation increased the pain threshold by a factor between 68% and 95% depending on the injected drug. We also observed a 54% reduction on the volume increase of the edema when it was irradiated. CONCLUSION: He-Ne LLLT inhibits the sensitization increase of nociceptors on the inflammatory process. The analgesic effect seems to involve hyperalgesic mediators instead of peripheral opioid receptors.

[Splanchnicectomy using thoracoscopy]. / Splanchnicectomie sous thoracoscopie.

A technique of thoracic splanchnicectomy under video thoracoscopic control is reported. This simple and non aggressive procedure is indicated for very painful forms of pancreatic cancer and for some cases of chronic pancreatitis. It should relieve pain for a longer period than splanchnic nerve injection or radiotherapy.