This review was not supported by grants. We thankSanypet S.p.a. (Padua, Italy) for kindly providing the dietary supplement and placebo used in this study. We also thank San Patrignano Community which kindly participated in the study by supplying all dogs.

This clinical evaluation was planned accordingly with the European legislation (86/609/EU) and the experimental protocol was approved by the Ethics and Welfare Animal Committee of the AIRMO Research Center (Milan, Italy).
1. Dogs and Food Selection
<ol>
	<li>Randomly divide 32 dogs of different breeds (7.01 &#177; 0.20 year [mean &#177; standard error of mean (SEM)]; 34.05 &#177; 1.47 Kg [mean &#177; SEM]; 12 males and 20 females) suffering from chronic halitosis into 2 groups, providing them with either the dietary supplement or the placebo over a period of 30 days.</li>
	<li>Ensure that each dog receives the appropriate dose of dietary supplement or placebo based on the animal&#8217;s weight in kilograms, according to the manufacturer&#8217;s instructions.&#160;(Table 1).</li>
	<li>After a 30 day treatment, ensure that both groups have a 10 day wash out, where all animals are fed with placebo. &#160;At the end of the wash out period, the feeding regimen for the two groups are reversed such that the control group receives the dietary supplement for another 30 days and the treatment group receives the placebo.</li>
	<li>Two veterinary inspections performed on the dogs in both groups, one before and one after the 30 day treatment.</li>
</ol>
2. The Gas Chromatograph
Note: The gas chromatograph is a portable device which measures the volatile sulphur compounds (VSC), hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, which are the major causative factors of halitosis, providing information about the concentration of each gas measured. Measuring each VSC helps to identify the cause of halitosis and assess treatment efficacy.
<ol>
	<li>Using the procedure outlined in sections 3 and 4, examine volatile sulphur compounds (VSC) before treatment with the dietary supplement (time 0), 10 days post-treatment (time 10), 20 days post-treatment (time 20) and at the end of treatment (time 30).</li>
</ol>
3. Gas Chromatograph Setting
<ol>
	<li>Turn on the main switch at the back of the unit. Note: The initial screen displays the word &#8220;MEASUREMENT MODE&#8221;.</li>
	<li>Press the ENTER key to select MEASUREMENT MODE. 
	Note: WAIT is displayed on the screen and the standby time required to stabilize the measuring device is displayed next to the word &#8220;COUNTER&#8221;. When the standby time has come to an end, the device is automatically ready to measure and the READY indicator lamp turns on.</li>
</ol>
4. Dogs Breath Sampling and Analysis
<ol>
	<li>Run the Data Manager software.</li>
	<li>Click &#8220;Measurement&#8221; after confirming that &#8220;Current status&#8221; is &#8220;Ready to measure&#8221;</li>
	<li>Enter ID and click &#8220;Select&#8221;. Note: A message is displayed at the upper right-hand corner of the window.
	<ol>
		<li>If the message displayed is &#8220;Inject oral gas&#8221;, start the measurement.</li>
		<li>If the message displayed is &#8220;The device is being ready&#8221;, then wait.</li>
	</ol>
	</li>
	<li>By means of a 1&#160;ml disposable syringe, collect the breath from the dog&#8217;s mouth by placing the syringe in the dog&#8217;s labial commissure, slowly pulling the plunger and removing the syringe from the dog&#8217;s mouth .</li>
	<li>Adjust the volume of the syringe to 1 ml.</li>
	<li>Attach the supplied needle to the end of the syringe and, after opening the lid, eject the sample into the inlet on the device main unit by pushing the plunger. 
	Note: Measurements start automatically and the READY indicator lamp goes off. Measurement is completed in 8 min and the result is automatically displayed (display can be changed to &#8220;Chromatogram&#8221;). The Judgment window appears upon completing the measurement. Measurement history and measured data can be displayed .</li>
	<li>Push ENTER to store the results into the unit. 
	Note: Data are expressed in standard units of parts per billion (ppb) and/or ng/10 ml.</li>
	<li>Then wait 1 min to stabilize the device and perform a new measurement. Note: At the end of this time, the lamp turns on meaning that the device is ready.</li>
</ol>

The authors have nothing to disclose.

Halitosis is a common problem in dogs, representing a psychosocial issue significantly affecting the pet-owner relationship19. Bad breath comes from microbial metabolism of both exogenous and endogenous protein substrates within the oral cavity20 which results in volatile sulphur compound production, and is worsened by other factors including acute necrotising ulcerative gingivitis, infected extraction site, debris under dental appliances, ulcers, tonsilloliths and low hygiene21-23. The main agents responsible for bad breath production are Gram-negative bacteria24 and their increase is related to a thickening of dental plaque25. Good oral hygiene along with specific diets can reduce the microbial load to the physiological level, reducing halitosis and avoiding gengivitis and periodontal diseases that have unpleasant effects on breath26.
This view is supported by our study showing that, after 10 days of dietary supplement intake, an overall improvement was observed in 29 out of 32 dogs (90%) presenting a moderateto-severe malodor and, at the end of the evaluation, 19 out of 29 dogs (65%) who achieved an improvement acquired a stabile physiological condition regarding VSCs concentration.
Our investigation broaches the very intriguing issue of the possible administration of a dietary supplement to dogs with breath malodor by basically introducing fish hydrolized proteins, sage, Ribes nigrum L., thyme, lysozyme, propolis, bioflavonoids and vitamin C in the daily nutritional schedule. These compounds, widely used in traditional medicine, have been already proven effective both in vitro and in vivo.
Specifically, fish proteins were chosen on the basis of previous chromatographic studies which pointed out the presence of great amounts of hydrogen sulfide, methanethiol, ethanethiol, dimethyl sulfide, and ethylene sulfide in beef proteins27. Literature studies have also shown an antimicrobial activity of sage and Ribes nigrum L. against Streptoccocus mutans which is known to be one of the oral pathogens (along with Porphyromonas gingivalis and Candida albicans) responsible for oral malodor and dental caries formation28-30. Bioflavonoids and vitamin C have been recognized as both antiinflammatory and bacteriostatic agents limiting the growth of certain bacteria associated with periodontal diseases31.
Regarding propolis and thyme, a reduction in malodor production from incubated whole saliva was demonstrated by Sterer et al.32 The bacteriostatic activity of propolis has been widely demontrated in literature, ranging from limiting the quantity of bacterial plaque33-37 to decreasing the tolerance of microorganisms to acid pH38, showing greater effectiveness than chlorhexidine gluconate solutions39. Moreover, propolis was proven effective in reducing teeth caries in rats limiting the number of microorganisms, slowing down synthesis of insoluble glucans, and glucosyltransferase activity40.
Our study presents some limitations such as the sensitivity of the instrument, if compared with other available techniques (e.g. bumpy WO3 hemitube nanostructure assisted by O2 plasma surface modification with functionalization of graphene-based material41, electrospun SnO2 nanofibers sensitized with reduced graphene oxide nanosheets42, benzoyl-DL-arginine-naphthylamide (BANA) test43, High Performance Liquid Chromatography (HPLC)44 and Solid-Phase Micro Extraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS45). On the other hand, the device employed in this study might allow, for instance, fast data collection to rapidly achieve an early diagnosis of viral hepatitis B, which is also characterized by elevated dimethile sulfide levels46 or to establish a state of systemic and/or respiratory disease (such as pneumonia, pulmonary emphysema and bronchitis) that are characterized by elevated hydrogen sulfide, methyl mercaptan and dimethyl sulfide levels47.
During the procedure we realized that the time available to collect the breath from a dog&#8217;s mouth was slightly lower than that required (few seconds against 30 sec suggested by the manufacturer). This problem was successfully overcome by repeating each measurement three times. A critical step during the experiment was breath collection. In order not to leave the dog with his mouth open for too long without damaging the syringe, we placed the syringe in the dog&#8217;s labial commissure in order to safely collect the breath.
As observed in humans, further applications of this technique will be the prevention of breath-related diseases, i.e. systemic and respiratory, in dogs and more generally in pets. In summary, our study showed that the dietary supplement was effective in reducing chronic halitosis in dogs. Our results might be also in agreement with what is proposed by Porter et al., who hypothesized a possible interplay between halitosis and the gastrointestinal tract suggesting that a therapy for halitosis with oral origin might also have some effect upon gut bacteria composition which is known to be responsible, in some cases, for volatile sulphur compounds production48.&#160;Nevertheless, a dysbiosis might also occur after a massive use of antibiotics49, or meat derived from intensive livestocks and commonly used in most of animal and human food50,51.&#160;

Oral malodor, also known by the term halitosis, is a common complaint which causes embarrassment in interpersonal social relationships1 as much as body malodor2. Although most epidemiological data on oral malodor is limited in accuracy and sensitivity due to a subjective self-estimation, studies report that 30-50% of the population is affected by this pathology3-6. Some studies have also evidenced that it is more prevalent in men than in women regardless of age with a ratio 3:1 and the ratio is slightly higher in people over 20 years7.
Halitosis can be staged depending on its etiology: type 1 (oral), type 2 (airway), type 3 (gastroesophageal), type 4 (blood-borne) and type 5 (subjective); however, it has been hypothesized that halitosis could be potentially considered the sum of these types in any combination, superimposed on the physiologic odour present in health (Type 0)8,9. Scully et al., who widely described and characterized aetiopathogenesis and management of halitosis, reported that in some patients it has an extra-oral aetiology and, in a few of them, metabolic anomalies are involved10. The authors pointed out that volatile sulphur compounds (VSC) and some predisposing factors, e.g. poor oral hygiene, hyposalivation, dental appliances, gingival and periodontal disease and mucosal disease, are mainly responsible for the malodor. Further, they proposed oral health improvement, anti-malodor therapies and malodor counteractives as a basic conventional approach. Moreover, they also proposed the use of probiotics and vaccines against malodor causing bacteria.
Human and animal companions, i.e. cats and dogs, can be affected by food-related adverse reactions which can involve several systems such as the gastroenteric, cutaneous, otological, ocular, urinary and respiratory systems11-13. However, several animal studies have pointed out that the nasal chamber is one of most involved systems where tartar14, stomatitis15, and halitosis16 are quite often present. Furthermore, bacteria that cause halitosis can be transmitted from animal companions to humans17. Iwanicka-Grzegorek quantified sulphur compounds and low molecular mass amines in saliva from 84 patients suffering from halitosis and 40 healthy controls. More than 80% of patients belonging to the halitosis group reported having pets in childhood (P &#60;0.001) and over 70% still owned a pet (P &#60;0.001).
Therefore nutrition can be used to manage halitosis. Moreover, nutrition plays a pivotal role in tooth development, oral and gum tissue integrity, bone resistance and even dental and oral disease management18. Based on such considerations, at the Italian Association for Oncological Research Disease (AIRMO) Center, we run&#160;a randomized placebo-controlled crossover clinical evaluation involving 32 dogs suffering from halitosis in order&#160;to investigate the efficacy of a dietary supplement vs. a commercially available pet food.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OralChroma CHM-1</td>
			<td>FIS Inc.</td>
			<td>10061285</td>
			<td>portable gas cromatograph</td>
		</tr>
		<tr>
			<td>FORZA10 Oral Active</td>
			<td>SANYpet .Sp.a.</td>
			<td></td>
			<td>dietary supplement</td>
		</tr>
		<tr>
			<td>InJ/Light-100 PCS</td>
			<td>Rays S.p.a.</td>
			<td>INJ5171112</td>
			<td>disposable syringes&#160;</td>
		</tr>
	</tbody>
</table>

therapeutic effectiveness of a dietary supplement for management of halitosis in dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

Both placebo and dietary supplement were adjusted in order to provide similar nutritional and caloric intake and to satisfy the nutritional requirement of adult dogs, in particular. Their analytical composition was: crude protein 26%, crude oils and fats 13%, crude fiber 2.5%, crude ash 8% and Metabolized Energy (EM) 3464 kcal/kg. Metabolized energy was calculated according to the Nutritional Guidelines for complete and complementary pet food for cats and dogs by The European Pet Food Industry Federation. Placebo was composed of dehydrated chicken meat, rice, animal fat,&#160;dehydrated eggs, oat, dry&#160;beet pulp, flax&#160;seeds, hydrolysed&#160;animal proteins, fish oil, vegetable oil, calcium carbonate, dry yeast beer, monocalcium phosphate, sodium chloride. Dietary supplement differed from the placebo for the presence of active substances contained in special heart-shaped cold pressed tablets (European patent n. EP 2526781) included in the complete food in a percentage of 6-7%, and for the protein source, fish meal instead chicken meal. Tablets contained propolis (0.0161%), Salvia officinalis (0.0087%), egg albumen (lysozyme 0.0078%), dehydrated orange extract (bioflavonoids 0.0077%), Thymus vulgaris (0.0127%), Ribes nigrum (0.0040%).
Data were analyzed, presented as the means &#177; standard error of the mean and checked for normality using the D&#8217;Agostino-Pearson normality test. A multiple t-test was used to compare changes in VSCs score at follow-up versus baseline for each treatment modality.
Figure 1A shows a significant decrease in hydrogen sulfide concentration in treated animals, from a baseline value of 0.43 ng/ml to 0.06 ng/ml, after 30 days treatment. Figure 1B shows a significant decrease of methyl mercaptan concentration in treated animals, from a baseline value of 0.30 ng/ml to 0.06 ng/ml, after 30 days treatment. Figure 1C shows a significant decrease in dimethyl sulfide concentration in treated animals, from a baseline value of 0.28 ng/ml to 0.13 ng/ml, after 30 days treatment.
Figure 2A shows a significant decrease in hydrogen sulfide concentration, from a baseline value of 0.37 ng/ml to 0.10 ng/ml, after 20 days treatment. In Figure 2B a similar trend is observed also for methyl mercaptan which shows a significant decrease of the compound concentration, from a baseline value of 0.24 ng/ml to 0.05 ng/ml, after 30 days treatment. As to dimethyl sulfide, Figure 2C, a significant decrease of the compound concentration, from a baseline value of 0.32 ng/ml to 0.11 ng/ml, was observed after 30 days treatment.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BODY WEIGHT (Kg)</td>
			<td>DIETARY SUPPLEMENT AMOUNT PER DAY (g)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 - 10</td>
			<td>30 - 180</td>
		</tr>
		<tr>
			<td>11 - 20</td>
			<td>190 - 300</td>
		</tr>
		<tr>
			<td>21 - 35</td>
			<td>310 - 455</td>
		</tr>
		<tr>
			<td>36 - 50</td>
			<td>465 - 595</td>
		</tr>
	</tbody>
</table>
Table 1: Daily Amount of Food Provided to Dogs.
<img alt="Figure 1" src="/files/ftp_upload/52717/52717fig1.jpg" /> 
Figure 1: (A) Graphical Representation of Hydrogen Sulfide Concentration Trend in Treated (n = 16) vs Control (n = 16) Group During the 30 Days Treatment Period; *p &#60;0.05; (B) Graphical Representation of Methyl Mercaptan Concentration Trend in Treated (n = 16) vs Control (n = 16) Group During the 30 Days Treatment Period; *p &#60;0.05; (C) Graphical Representation of the&#160;Dimethyl Sulfide Concentration Trend in Treated (n = 16) vs Control (n = 16) Group During the 30 Days Treatment Period; *p &#60;0.05.
<img alt="Figure 2" src="/files/ftp_upload/52717/52717fig2.jpg" /> 
Figure 2: (A) Graphical Representation of Hydrogen Sulfide Concentration Trends in Treated (n = 16) vs Control (n = 16) Group after the 10 Days Wash Out Period; *p &#60;0.05; (B) Graphical Representation of Methyl Mercaptan Concentration Trend in Treated (n = 16) v.s. Control (n = 16) Group after the 10 Days Wash Out Period; *p &#60;0.05; (C) Graphical Representation of Dimethyl Sulfide Concentration Trends in Treated (n = 16) v.s. Control (n = 16) Group after the 10 Days Wash Out Period; *p &#60;0.05.

Watch this Scientific Journal Video about Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs at JoVE.com

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

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11.3K Views.  Azienda Ospedaliero-Universitaria Policlinico di Modena. We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

Video: Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise function and quality of life. Moreover, clinical practice suggests that, unlike other inflammatory myopathies, the majority of IBM patients are not responsive to treatment with immunosuppressive or immunomodulatory drugs to counteract disease progression1. Additionally, conventional resistance training programs have been proven ineffective in restoring muscle function and muscle mass in these patients2,3. Nevertheless, we have recently observed that restricting muscle blood flow using tourniquet cuffs in association with moderate intensity resistance training in an IBM patient produced a significant gain in muscle mass and function, along with substantial benefits in quality of life4. Thus, a new non-pharmacological approach for IBM patients has been proposed. Herein, we describe the details of a proposed protocol for vascular occlusion associated with a resistance training program for this population.

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Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
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Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...