Short term treatment of secondary lymphedema with hyaluronidase injections reduces mouse hindlimb lymphedema.

Lymphedema is a common complication following breast cancer treatment with axillary lymphadenectomy and radiotherapy. Currently, there is no curative treatment for this disease, hence there is a need for new therapeutic suggestions. The aim of this study was to investigate the effect of hyaluronidase (HYAL) injections after inducing hindlimb lymphedema in 36 female C57BL/6 mice. HYAL injections were administered every second day for 14 days in three groups: (1) HYAL for 1 week followed by saline for 1 week, (2) HYAL for 2 weeks, and (3) saline injections for 2 weeks. Volume of the lymphedema limb was weekly assessed with micro-computed tomography (µ-CT) scans for a total course of 6 weeks. Lymph vessel morphometry was assessed in the end of the study after staining cross-sections of the hindlimb for anti-LYVE-1 blindly. Lymphatic function was assessed by lymphoscintigraphy to assess lymphatic clearance. There was a significant reduction of the volume of lymphedema in mice treated with HYAL-7 compared with mice treated with HYAL-14 (p < 0.05) and saline (p < 0.05). No differences were detected in lymph vessel morphometry and the lymphoscintigraphy between groups. Short-term treatment with HYAL-7 might be a potential therapeutic suggestion for secondary lymphedema induced in mouse hindlimbs. In the future, clinical studies are needed to investigate the potential of HYAL treatment in human beings.

Comparison between stromal vascular fraction and adipose derived stem cells in a mouse lymphedema model.

Background: Lymphedema is one of the most common complications following breast cancer. Axillary lymph node dissection and radiotherapy are two well-known risk factors resulting in either removal or damage to the lymph nodes. As stem cells are known for their regenerative capabilities, they could theoretically repair/restore the damaged lymph vessels leading to a decrease in lymphedema.Methods: We evaluated the treatment of SVF and ASC on a mouse lymphedema model. Forty-five mice were allocated into three groups containing 15 mice each. The SVF group was injected with 100 µl containing 1 × 106 SVF, the ASC group with 100 µl ml containing 1 × 106 ASC and the NS with 100 µl ml of NS. Volumes of the mice were assessed weekly by µCT hindlimb volumetry for a total of 8 weeks. Lymph vessel morphometry was assessed by cross-sections of both hindlimbs stained for anti-LYVE1. Lymphatic function was assessed by lymphatic clearance.Results: The volume change between the groups was non-significant throughout all 8 weeks. The immunohistochemistry showed a statistically significant difference between the hindlimbs in ASC vs. NS group p = 0.032, 95% CI [-2121, -103].Conclusion: The volume of the hindlimbs showed that treatment with SVF or ASC yielded very similar results compared to the control group when assessed after 8 weeks. In week two the biggest difference between ASC and NS was seen but the difference diminished during the 8 weeks. The secondary outcomes showed that the lymph vessel lumen decreased when treated with ASC compared to the control group. Lymphoscintigraphy yielded non-significant results.

Quantification of Chronic Lymphedema in a Revised Mouse Model.

BACKGROUND: Lymphedema is a common and debilitating complication following cancer treatment with surgical lymph node excision and radiotherapy. Currently there are no curative treatments for lymphedema. Animal models that intended to replicate the disease have been inadequate, making a troublesome transition from experimental therapeutic studies into the clinic. It is therefore imperative to establish an experimental animal model that can reliably replicate clinical lymphedema. METHODS: To discover the optimal method of lymphedema induction, surgical lymph ablation and irradiation or silicone splint emplacement were combined in 8 experimental groups (n = 4). In total, 32 mice served in this study and were followed for 8 weeks after surgery. Outcomes included micro-computed tomography hind limb volumetry, lymphatic clearance measured with technetium Tc 99m (Tc) human serum albumin lymphoscintigraphy and lymph vessel ectasia quantified with LYVE-1 immunohistochemistry. RESULTS: All trialed models but one resulted in only transient lymphedema or lasting lymphedema with adverse morbidity. Combined surgical lymph obstruction with 2 fractions of 10-Gy irradiation successfully induced lasting lymphedema without adverse events. Over the 8 weeks' follow-up, limb volumes were significantly increased at all time points (P < 0.001), lymph drainage was impaired (P < 0.001), and lymph vessels were ectatic (P < 0.001), when compared with the unoperated limbs. CONCLUSIONS: The presented model of acquired lymphedema is a reduction and refinement of previous works and can transpose to future observational and interventional studies. In addition, it is shown how Tc-HSA lymphoscintigraphy can quantify lymphatic clearance, which can prove insightful in therapeutic studies aiming to enhance lymphatic drainage.

A Scintigraphic Method for Quantitation of Lymphatic Function in Arm Lymphedema.

BACKGROUND: Lymphoscintigraphy is commonly used to assess breast cancer-related lymphedema. However, a reliable quantitative method that clearly distinguishes normal lymphatic function from lymphedema is desired. We propose a quantitative method based upon the physiological mean transit time (MTT) measure of lymph fluid passing through the arm. METHODS AND RESULTS: Eleven patients, aged 34-68 years, with unilateral arm lymphedema following breast cancer treatment underwent simultaneous bilateral lymphoscintigraphy using intradermal injection of 99mTc-labeled human serum albumin (HSA). Imaging was performed at 30-45 minute intervals for 5 hours. Time activity curves from each injection site and each arm region were recorded. The input into the arm region was obtained as the (minus) time derivative of the injection site activity curve. In the proposed model the arm activity curve was considered to arise from the convolution of the retention function and the input function. The retention function was obtained by fitting the calculated arm activity curve to the measured arm activity curve. The MTT of activity passing through the arm was calculated as the time integral of the resulting retention function. All measured time activity curves were well described by the model. The MTT of the lymphedema arm (mean 60.1 minutes, range 22-105 minutes) was markedly different from that of the contralateral normal arm (mean 5.4 minutes, range 1.2-8.7 minutes), p < 0.0001. CONCLUSION: The proposed model showed great similarity with measured time activity curves and was capable of quantitatively distinguishing lymphatic function of the lymphedema arm from that of the normal arm in terms of calculated MTT.

Tc-99m-Human Serum Albumin Transit Time as a Measure of Arm Breast Cancer-Related Lymphedema.

BACKGROUND: Lymphoscintigraphy has often been used for evaluating arm lymphatic dysfunction, but no optimal approach for quantification has so far emerged. We propose a quantifiable measure of lymphatic function that we applied in patients treated for breast cancer. METHODS: Eleven patients, aged 34-68 years, with unilateral arm lymphedema following breast cancer treatment underwent bilateral lymphoscintigraphy using intradermal injection in both hands of technetium-99m-labeled human serum albumin and sequential 5 min imaging for 5 hours. The mean transit time (MTT) in the arms was calculated based on time activity curves generated from injection site and arm regions. Visual lymphedema scoring was performed based on dermal backflow and lymph node presence. Excess arm volume was calculated from circumference measurements. RESULTS: The MTT (mean ± SD) was significantly longer in the lymphedema arm than in the normal arm: 60.1 ± 27.7 versus 5.4 ± 2.5 minutes (mean difference, 54.7 minutes; 95% confidence interval, 36.5-72.9 minutes; P < 0.0001). Patients with previous erysipelas infection had significantly longer MTT than other patients (mean difference, 43.7 minutes; 95% confidence interval, 18.6-68.7 minutes; P < 0.001). There was a positive correlation between MTT and excess arm volume (r = 0.64; P = 0.04) and number of lymph nodes removed (r = 0.65; P = 0.03) but no correlation between visual score and MTT. CONCLUSION: Measurements of MTT were able to discriminate lymphedema from healthy arm and MTT correlated with relevant markers for lymphedema severity. We encourage further research using the MTT approach for monitoring lymphedema and evaluation of treatment response.

Conditional ablation of myeloid TNF increases lesion volume after experimental stroke in mice, possibly via altered ERK1/2 signaling.

Microglia are activated following cerebral ischemia and increase their production of the neuro- and immunomodulatory cytokine tumor necrosis factor (TNF). To address the function of TNF from this cellular source in focal cerebral ischemia we used TNF conditional knock out mice (LysMcreTNF(fl/fl)) in which the TNF gene was deleted in cells of the myeloid lineage, including microglia. The deletion reduced secreted TNF levels in lipopolysaccharide-stimulated cultured primary microglia by ~93%. Furthermore, phosphorylated-ERK/ERK ratios were significantly decreased in naïve LysMcreTNF(fl/fl) mice demonstrating altered ERK signal transduction. Micro-PET using (18)[F]-fluorodeoxyglucose immediately after focal cerebral ischemia showed increased glucose uptake in LysMcreTNF(fl/fl) mice, representing significant metabolic changes, that translated into increased infarct volumes at 24 hours and 5 days compared to littermates (TNFfl/fl). In naïve LysMcreTNF(fl/fl) mice cytokine levels were low and comparable to littermates. At 6 hours, TNF producing microglia were reduced by 56% in the ischemic cortex in LysMcreTNF(fl/fl) mice compared to littermate mice, whereas no TNF(+) leukocytes were detected. At 24 hours, pro-inflammatory cytokine (TNF, IL-1ß, IL-6, IL-5 and CXCL1) levels were significantly lower in LysMcreTNF(fl/fl) mice, despite comparable infiltrating leukocyte populations. Our results identify microglial TNF as beneficial and neuroprotective in the acute phase and as a modulator of neuroinflammation at later time points after experimental ischemia, which may contribute to regenerative recovery.

Cardiac abnormalities assessed by non-invasive techniques in patients with newly diagnosed idiopathic inflammatory myopathies.

OBJECTIVES: Knowledge of cardiac involvement in idiopathic inflammatory myopathies (IIM) is limited, especially in the early stage of disease. The objective of the present study was to perform a controlled evaluation of cardiac abnormalities in newly diagnosed, untreated patients with idiopathic inflammatory myopathies (IIM) by means of non-invasive techniques. METHODS: Fourteen patients with IIM (8 polymyositis, 4 dermatomyositis, 2 cancer-associated dermatomyositis) and 14 gender- and age- matched healthy control subjects were investigated. Participant assessments included a cardiac questionnaire, cardiac troponin-I (TnI), electrocardiogram (standard 12-lead and 48-h Holter monitoring), echocardiography with tissue Doppler measures, cardiac magnetic resonance (CMR) imaging with T2 mapping and semi-quantitative (99m)technetium pyrophosphate ((99m)Tc-PYP) scintigraphy. RESULTS: Dyspnoea was present in 8 (57%) of the patients compared to none of the controls (p<0.01). Median levels of TnI in patients and controls were 20 ng/L and 6 ng/L, respectively (p=0.06). QTc intervals were prolonged in the patient group (p=0.01). Two patients had systolic dysfunction, and one diastolic dysfunction. The myocardial (99m)Tc-PYP uptake and CMR results differed between patients and controls, albeit not with statistical significance. Overall, cardiac abnormalities were demonstrated in 9 (64%) of the patients versus 2 (14%) of the controls (p=0.02). CONCLUSIONS: Cardiac abnormalities assessed by TnI, ECG or imaging modalities were significantly more common in newly diagnosed, treatment naïve patients with IIM compared to healthy control subjects. These abnormalities, although subclinical, may indicate that myocardial involvement is common in patients and calls for larger controlled studies and further investigations of the prognostic implications of this finding.

Estimation of Tumor Volumes by 11C-MeAIB and 18F-FDG PET in an Orthotopic Glioblastoma Rat Model.

UNLABELLED: Brain tumor volume assessment is a major challenge. Molecular imaging using PET may be a promising option because it reflects the biologically active cells. We compared the agreement between PET- and histology-derived tumor volumes in an orthotopic glioblastoma rat model with a noninfiltrating (U87MG) and an infiltrating (T87) tumor phenotype using 2 different radiotracers, 2 different image reconstruction algorithms, parametric imaging, and 2 different image segmentation techniques. METHODS: Rats with U87MG- and T87-derived glioblastomas were continuously scanned with PET for 1 h starting immediately after the injection of 11C-methylaminoisobutyric acid (11C-MeAIB). One hour later, 18F-FDG was injected, followed by a 3-h dynamic PET scan. Images were reconstructed using 2-dimensional ordered-subsets expectation maximization and 3-dimensional maximum a posteriori probability (MAP3D) algorithms. In addition, a parametric image, encompassing the entire tumor kinetics in a single image, was calculated on the basis of the 11C-MeAIB images. All reconstructed images were segmented by fixed thresholding of maximum voxel intensity (VImax) and mean background intensity. The agreement between PET- and histology-derived tumor volumes and intra- and interobserver agreement of the PET-derived volumes were evaluated using Bland-Altman plots. RESULTS: By PET, the mean U87MG tumor volume was 35.0 mm3 using 18F-FDG and 34.1 mm3 with 11C-MeAIB, compared with 33.7 mm3 by histology. Corresponding T87 tumor volumes were 122.1 mm3 using 18F-FDG, 118.3 mm3 with 11C-MeAIB, and 125.4 mm3 by histology. None of these volumes were significantly different. The best agreement between PET- and histology-derived U87MG tumor volumes was achieved with 11C-MeAIB, MAP3D reconstruction, and fixed thresholding of VImax. The intra- and interobserver agreement was high using this method. For T87 tumors, the best agreement between PET- and histology-derived volumes was obtained using 18F-FDG, MAP3D reconstruction, and fixed thresholding of mean background intensity. The agreement using 11C-MeAIB, parametric imaging, and fixed thresholding of VImax was slightly inferior, but the intra- and interobserver agreement was clearly superior. CONCLUSION: Estimation of tumor volume by PET of noninfiltrating brain tumors was accurate and reproducible. In contrast, tumor volume estimation by PET of infiltrating brain tumors was difficult and hard to reproduce. On the basis of our results, PET evaluation of highly infiltrating brain tumors should be further developed.